Ligand-dependent corepressor (LCOR) variants and their use

Abstract

The present invention provides novel and improved cancer therapies based on isolated or synthesized polynucleotide sequences encoding ligand-dependent corepressor (LCOR) variants or fragments thereof that can induce the expression of antigen processing / presentation mechanism (APM) genes, wherein the ligand-dependent corepressor (LCOR) variant or fragment comprises a functional HTH domain, and further comprises either a nuclear receptor-binding domain that is absent or an inactivated nuclear receptor-binding domain.

Description

[Technical Field]

[0001] This invention relates to the field of immunotherapy, more specifically to cancer immunotherapy or a treatment method that uses the action of the immune system to eliminate cancer cells. [Background technology]

[0002] Immunotherapy with immune checkpoint blockers (ICBs) is an immunotherapy that induces an anti-tumor immune response and has emerged as one of the most effective treatments in oncology. Nevertheless, most patients are ineffective with ICBs or develop resistance to them, and this strategy is not effective against all cancer types. Therefore, the endogenous tumor mechanisms underlying ICB resistance are still being actively studied. Among these, interferon sensitivity in tumor phenotypic heterogeneity may help explain how patients with similar tumor types nevertheless exhibit different sensitivities to ICBs.

[0003] Interferon signaling plays a central role in tumor immunosurveillance and immunotherapy responses, partly through the modulation of antigen presentation mechanisms (APMs) in tumor cells. APMs include, among others, major histocompatibility complex (MHC) class I genes (HLA), accessory molecules (β2M), transporters (TAP complexes and tapasin), and immunoproteasome genes (PSMBs), which are intracellular effectors of antigen presentation that enable the immune system to recognize antigens. Multiple studies and clinical reports have identified genetic alterations in antigen presentation components and the IFN pathway as major mechanisms of ICB resistance. Epigenetic factors and transactivators, such as EZH2, NF-κB, and NLRC5, can also modulate MHC-I and other APM genes and influence tumor immunogenicity. Therefore, a better understanding of APM modulation in different tumor cell populations and tumor phenotypes may help elucidate the underlying mechanisms of immunotherapy resistance.

[0004] Stem cell phenotypes, such as embryonic stem cells and slow-cycle adult stem cells, downregulate MHC and aid in evading immune surveillance. Cancer stem cells (CSCs) resemble their normal counterparts in many respects and may utilize similar immune-evading phenotypes associated with ICB resistance. CSCs actively influence each other through immunosuppressive cytokines, including IFN, and several reports indicate low expression of MHC and transporter 1 (TAP1) in CSCs, suggesting a role in evading immune surveillance. CSCs also express CD274 (PD-L1), a mechanism that contributes to immunosuppression but not to evading anti-PD-L1 ICBs. Epithelial-mesenchymal transition, often associated with stem cell characteristics, also promotes immunosuppressive features. In the context of immunotherapy resistance, tumor progenitor cells (TICs) of cutaneous squamous cell carcinoma (SCC) are associated with adoptive cell migration resistance due to upregulation of CD80, while CSCs of head and neck SCC exhibit ICB resistance through downregulation of T cell-mobilizing chemokine secretion. However, the relationship between stem cell characteristics and APM pathway dysregulation leading to ICB escape, and the molecular mechanisms regulating this, have not been established.

[0005] In the mammary gland, normal mammary stem cells (MaSCs) and CSCs are typically governed by developing cell fate determinants and stem cell transcription factors. LCORs are differentiation factors that enhance the sensitivity of these cells to IFN, driving endogenous tumor cell differentiation and leading to reduced tumor growth. However, how LCORs interact with the IFN response has not been investigated and may be important for understanding cellular immunity. Therefore, we were interested in investigating their mechanistic relationship with immunity and ICB resistance in triple-negative breast cancer (TNBC). This is because, to date, there has been limited clinical research in TNBC. There is an urgent need to improve the efficacy of ICB in breast cancer, which has only shown efficacy so far, and therefore it has clinical significance. In this specification, the inventors of LCOR lowWe demonstrate that breast CSCs suspend antigen processing and presentation, thereby contributing to immune checkpoint therapy resistance in TNBCs. LCORs, independently of IFNs, activate APM transcription, thereby making CSCs visible in ICBs and vulnerable to immune attack. Our results demonstrate the relevance of LCOR biology in phenotypic heterogeneity and APM regulation as an excellent therapeutic partner for ICB therapy. [Brief explanation of the drawing]

[0006] [Figure 1]a: Figure of growth curves of 4TO7 tumors in Balb / c mice treated with anti-PD-L1 ICB. Dose and regimen are shown. n = 3 tumors per condition. b: Figure of GO analysis by RNA-seq of upregulated (red) and downregulated (blue) pathways in 4TO7 anti-PD-L1 IRT and control cells (I-Ctrl). P values ​​were calculated by Fisher exact test. c: Figure of GSEA of APM (GO:0048002) in IRT vs. control cells. Family-wise error rate (FWER) P values ​​and FDR q values ​​are shown. d: Figure of GSVA for the following previously described stem cell signatures: embryonic stem cells (ES_1), NANOG-OCT4-SOX2 targets (NOS_Targets), and breast_CSCs (LIU breast ER-CSCs). Circle size represents significance, and color represents row z score mean. b~d: Two independent biological replicates. e: Figure of RT-qPCR of stem cell genes and differentiation genes in IRT vs. I-Ctrl tumor cells. Log2 factor change between IRT and control cells. Data represent mean ± standard error of the mean. f: Figure of flow cytometry analysis of CD24hi / CD29hi population in IRT and control cells. Data represent mean ± standard error of the mean. e, f: n=3 individual biological replicates. g: Figure of MFP injection and LDA of IRT 4TO7 cells in NSG mice. The table shows serial dilution injections along with engraftment rates, and n indicates the number of MFP injections. TIC frequency calculated by ELDA software is shown in red. P-values ​​were obtained by Pearson's chi-squared two-tailed test. h: Figure of CTL assay and CD24hi / CD44hi CSC flow cytometry analysis. Data represent the percentage of viable cells in a representative experiment. 1 μg / ml anti-PD-L1 treatment. i: Figure of the OT-I / JEDI CTL assay using CSCs and non-CSCs isolated from AT3-OVA and 4TO7-EGFP cells. E: T represents the effector versus target ratio. h, i: Data represent mean ± standard error of the mean. n = 3 independent biological replicates. j, k: Figure of the GSEA of the transcriptome-ranked list of responders versus non-responders in the TONIC trial 40, using our IRS(j) and ES_1(k) and LIU breast ER-CSC signatures.e, i: Accurate p-values ​​by two-tailed Student's t-test; d: Wilcoxon signed-rank test. f, h, i: Cells were gated from P3. [Figure 2]a: Figure showing isolation of PDO and CD24lo / CD44hi (CSC) and CD24hi / CD44lo (non-CSC) from four TNBC patients. Right: RT-qPCR analysis of LCOR and APM genes. n=4 independent biological replicates; data represent mean ± standard error of mean. b: Figure showing flow cytometry analysis of pan-HLA and β2M in PDO-derived CSC and non-CSC cells. n=3 independent biological replicates. Data represent relative mean fluorescence intensity (MFI; MFI relative to isotype control) ± standard error of mean. c: Figure showing representative LSFM imaging of PDO with CSC SORE6-GFP reporter (green), pan-HLA-ABC (purple), and DAPI (blue). The right panel shows single channels. n=3 independent organoids. d: Figure showing flow cytometry analysis of LCOR-GFP knock-in in MDA-MB-231 cells. e: Figure of RT-qPCR analysis of the APM gene in LCOR knock-in MDA-MB-231 cells isolated for each LCOR-, LCORlow / med, and LCORhigh. d, e: n=3 individual biological replicates; data represent mean ± standard error of the mean. f: Figure of pan-HLA-ABC in LCOR knock-in MDA-MB-231 cells: LCOR-GFP (green), HLA-ABC (purple), and DAPI (blue). n=3 independent biological replicates; image of one representative experiment. g, h: Figures of flow cytometry isolation (g) and RT-qPCR analysis (h) of OVA- / low and OVAhigh (SIINFEKL OVA peptide) from AT3 cells showing ectopic OVA overexpression (AT3-OVA). g, h: n=3 individual biological replicates; data represent mean ± standard error of the mean. i: This figure shows the flow cytometry analysis of the distribution of OVA- / low and OVA-high cells within the CD24lo / CD44hiCSC marker in AT3 cells. j: This figure shows orthotopic MFP injection and LDA of OVA- / low and OVA-high AT3-OVA cells into NSG immunodeficient mice. The table shows serial dilution injections along with the corresponding engraftment rates, and n indicates the number of MFP injections in each dilution. TIC frequencies calculated by ELDA software are shown in red. P-values ​​were obtained by Pearson's chi-squared two-tailed test.c, f: Scale bars are 100 μm and 40 μm, respectively. a, b, h: Exact P values ​​determined by two-tailed Student's t-test; e: *P<0.05, **P<0.01, ***P<0.005 (by one-way ANOVA and Bonferroni post-hoc test). a, b, e, g: Cells were gated from P3. [Figure 3]a: This figure shows the correlation between LCOR mRNA and APM (KEGG:M16004) signatures in 39 ER-negative breast cancer cell lines from CCLE. P-values ​​were calculated using low correlation. b: This figure shows the GSEA analysis using APM (KEGG:M16004) signatures from the METABRIC TNBC dataset, stratified as LCOR-high vs. LCOR-low patients based on median LCOR. FWER P-values ​​and FDR q-values ​​are shown. c, left: This figure shows the GSEA analysis of our ICB resistance signatures in the TNBC METABRIC dataset, stratified by LCOR median. Right: This figure shows the correlation between GSVA scores and LCOR for ICB resistance signatures in the METABRIC TNBC dataset. The blue line represents linear regression analysis using low correlation and P-values. d: This figure shows the heatmap of RT-qPCR analysis of the APM gene compared to controls in MDA-MB-231 cell lines transduced to LCOR-OE and LCOR-KD, respectively. n=3 individual biological replicates; data represent mean values. e~g: Figures of flow cytometry analysis of TAP1(e), β2M(f), and pan-HLA(g) levels in MDA-MB-231 LCOR-OE and LCOR-KD cells compared to their controls and isotype-negative controls under basic conditions, 10 ng / ml IFNγ treatment, and ruxolitinib (1 μM). n=3 independent biological replicates; data represent MFI ± standard error of mean value for isotype. h: Figure of flow cytometry analysis of SIINFEKL OVA peptide presented by H2-K1b in AT3-OVA cells under indicated conditions (10 ng / ml IFNγ treatment and 1 μM ruxolitinib). n=3 independent biological replicates; data represent MFI ± standard error of mean value for isotype. d~h: Exact P values ​​calculated by two-sided Student's t-test. e~h: Cells were gated from P3. [Figure 4]a: Figure of ChIP-seq analysis of pLEX-HA, pLEX-LCOR-HA, pLEX-LSKAA-HA, and pLEX-ΔHTH-HA (helix-turn-helix) in MDA-MB-231 cells. LSKAA is an LCOR variant with two amino acid changes in this domain that disrupt the proper interaction of the nuclear receptor domain with the nuclear receptor. Genome-wide distribution of the LCOR-HA peak and the LCOR-HA peak in chr6 under the conditions shown. Two independent biological replicates. b: Figure of heatmap RT-qPCR analysis of the APM gene located in the MHC cluster of chr6 in MDA-MB-231 cells, represented by log2 magnification change (log2(FC)). n=3 individual biological replicates; data represent mean values. c: Figure of ChIP-seq occupancy of the APM gene in this particular region of chr6 and β2M on chr15. Horizontal blue shading indicates either H3K27ac or H3K4me3 GRE as defined by UCSC (https: / / genome.ucsc.edu / ). Occupancy and scale are shown. Data show a reproduction. d: Figure of gene pathway analysis showing LCOR ChIP-seq peaks using BioCarta-2016. Data represent -log10(P); P values ​​calculated from Fisher's exact test. e: Figure of ChIP-seq HOMER analysis and the top 5 enriched binding motifs of LCOR peaks in MHC cluster regions. P values ​​of enrichment and percentage (%TP) of motif sequences labeled and classified as positive among the peaks. P values ​​calculated from Fisher's exact test. f: Schematic diagram of an interferon-stimulated modulating element (ISRE)-red fluorescent protein (RFP) reporter containing six tandem ISRE motifs and a minimal CMV promoter (mCMVp). g: Figure of flow cytometry analysis normalized to control conditions using ISRE and ISRE mutant reporters in MDA-MB-231 cells under the indicated conditions. n = 3 individual biological replicates; data represent standard error of MFI ± mean relative to isotype control mean.h: Figure of flow cytometry analysis of ISRE reporter in LCOR-GFP knock-in MDA-MB-231 under control (ctrl) conditions and after 24-hour treatment with IFNγ (10 ng / ml), ruxolitinib (0.5 μM), or BX-795 (TBK1i, 0.5 nM). Left: mCherry density plots and MFI in LCOR-, LCORmed / low, and LCORhigh. Right: MFI quantification. n=3 individual biological replicates; data represent relative MFI mean ± standard error of mean. g, h: Exact P values ​​calculated by one-way ANOVA and Bonferroni post-hoc analysis; cells were gated from P3. [Figure 5]a-e: Figures of cytotoxic lymphocyte (CTL) assays of AT3-OVA cells after 72 hours of co-culture with OT-1 CD8+ T cells under the indicated conditions and ratios of effector (E) cells to tumor (T) cells. a: Lcor-OE vs. control vector. b: Figure of time-lapse confocal imaging (1:1 ratio) of a. Tumor cells are labeled red, CD8+ T cells are labeled blue, and the cell death tracker (Sytox) is labeled green (shaded in white when mixed with tumor cells). Blue arrows indicate tumor immune interactions, and white arrows indicate tumor cell killing. c: Figure of Lcor-OE siRNA β2m vs. Lcor-OE siRNA control. d: Figure of Lcor-KD vs. control vector. e: Figure of flow cytometry analysis of CD69 expression in CD8+ T cells after CTL. a-e: n=3 independent biological replicates; a, c, d: data represent mean ± standard error of mean; e: relative MFI of representative replicate trials. f: Figure of quantitative analysis of IHC and percentage of tumor-infiltrating immune cells (CD45+) and CD8+ T cells (CD8+) in control vs. Lcor-OE 4TO7 tumors. n=3 individual biological replicates; data represent mean ± standard error of mean. g: Figure of flow cytometry analysis of percentage of tumor-infiltrating lymphoid cells (CD45+CD3+), CD8+ and CD4+ T cells in control or Lcor-OE 4TO7 tumors. n=10 individual biological replicates; data represent mean ± standard error of mean. h, i: Figure of computer clustering of immunophenotypes by deconvolution of the TNBC METABRIC dataset using xCell50. h: Heatmap representing patient clusters based on the four IPs shown. i: LCOR levels in each immunophenotype. b, f: Scale bar is 20 μm. a, c, d, g, h: Accurate p-values ​​by two-tailed Student's t-test; i: One-way ANOVA and Bonferroni post-hoc analysis. [Figure 6]a: Figures showing immunohistochemistry (IHC) analysis of LCOR in patient-derived samples before and after treatment for TNBC. P1 and P2 were treated with anti-PD-L1 atezolizumab + Abraxane and carboplatin, while P3 and P4 were treated with anti-PD1 nivolumab + SYK / FTL3 inhibitor (TAK-659). Left: LCOR nIRS in pre-treatment vs. post-treatment samples. Right: Representative images of pre-treatment and post-treatment samples (LCOR nIRS=6 and nIRS=2, respectively). P-values ​​by Wilcoxon signed-rank test. b, c: Figures showing analysis of LCOR mRNA levels in patients who responded to all induction therapy (CR+PR) versus patients who did not respond (PD+SD) in the TONIC trial (b) and I-SPY2 trial (c), comparing patients who responded to all induction therapy (CR+PR) versus patients who did not respond (PD+SD), comparing patients who responded to all induction therapy (CR+PR) and patients who showed disease progression (SD+PD). n is shown separately for each condition; data in both represent mean ± standard error of the mean. d: Figure of growth curve of orthotopically transplanted 4TO7 tumors and individual treatments under the indicated conditions, starting from the tumor size of 0.5 × 0.5 cm². Treatment was administered intraperitoneally weekly with the indicated dosing regimen. n = 10 mammary glands for each condition; data represent mean ± standard error of the mean. e: Figure of waterfall plots showing the percentage change in tumor volume from the first treatment day to each endpoint. n = 10 mammary glands for each condition. f: Figure of complete response (CR) rate to treatment and tumor-free mouse survival rate 12 months after discontinuation of treatment. This is a summary of 5 independent experiments (n=10 per experiment) treated with anti-PD-L1 (10 mg / kg) every 3 to 7 days. g: Figure of carmine staining of representative healed mammary glands. n = 3 healed mammary glands. h: Figure of growth curves of orthotopic 4TO7 control versus Lcor-KD tumors with the indicated dosing regimen every 3 days. n=10 mammary gland tumors; data represent mean ± standard error of the mean. i: Figure of preclinical lung metastasis assay by TV infusion of 4TO7 cells. Once metastasis was established, anti-PD-L1 treatment with the indicated dosing regimen was initiated. n=6 mice per condition; data represent mean ± standard error of the mean on a log10 scale. Veh.: Vehicle. b, c: Accurate P-values ​​by one-sided Student's t-test; h, i: One-way ANOVA. NT: Untreated (d, h, i). [Figure 7]a: Figure showing Lcor mRNA levels in EVs derived from HEK293T cells exhibiting ectopic overexpression of Lcor-HA (pLEX-Lcor-HA) versus control (pLEX-HA). Data represent the mean ± standard error of the mean across three technical replicates. Representative experiment from five independent experiments listed in the source data. b: Figure showing in vitro treatment of control and Lcor-HA-EVs over 3 days and anti-HA immunoblotting in 4TO7 cells. Representative immunoblotting from two biological replicates. c: Figure showing Lcor-HA protein expression and localization in recipient cells. Representative image from three independent biological replicates. d: Figure showing EV delivery, uptake, and protein translation in vivo: quantification of IHC and nuclear positivity in metastatic tumor tissue with EV posterior orbital administration and anti-HA. n=3 independent biological replicates; data represent the mean ± standard error of the mean. e: Figure showing preclinical lung metastasis assay by TV injection of 50,000 4TO7 cells. Metastasis was established after 8 days, and EV+anti-PD-L1 treatment with the indicated regimen was initiated and repeated every 3 days. n = 6 mice per condition; data represent mean ± standard error of the mean on a log10 scale. Representative BLI images (left) and growth curves (right) for the indicated conditions. f: Figure of Kaplan-Meier mouse survival rate plot for the lung metastasis assay of e. Tx: Treatment. P-values ​​were calculated by log-rank test. g: Figure of representative images of lung metastases established and treated with control EV+anti-PD-L1 (left), Lcor EV (center), and Lcor EV+anti-PD-L1 (right). h: This is a schematic diagram of the following main points of this study: (1) LCORlowCSCs resulted in immunoediting evasion and resistance to ICB treatment in TNBCs; (2) LCOR increased APM gene expression via ISRE independently of IFN; (3) LCOR-mediated immunogenicity influenced the response to ICB, CSC removal, and overcoming resistance in TNBCs; and (4) LCOR mRNA therapy resulted in enhancement of APM in CSCs, preventing their evasion to ICB, thereby leading to complete pathological response and cure. c, d: Scale bar, 60 μm; g: 300 μm.d: Accurate p-value obtained by a two-tailed Student's t-test; e: One-way analysis of variance; f: Log-rank test. [Figure 8] This figure shows the results of a 5-day cytotoxic lymphocyte (CTL) co-culture assay of human MCF7, estrogen-positive breast cancer cells, with and without anti-PD-L1 (atezolizumab), using human peripheral blood mononuclear cells (PBMCs). n=3 individual biological replicates; data represent mean ± SEM. MCF7 cells were transduced to ectopically overexpress the indicated types of LCOR, including: LCOR (LCOR wild-type); LSKAA (LCOR with two amino acid changes in the nuclear receptor domain); and delta-HTH (ΔHTH; LCOR without the HTH domain). [Figure 9] This figure shows a heatmap of the APM gene located in the MHC cluster of chr6 in MCF7 (ER+ breast cancer cells) and MDA-MB-231 (ER-negative breast cancer cells) as represented by log2 magnification change (log2(FC)) during RT-qPCR analysis. n=3 individual biological replicates; data represent mean values. [Figure 10] This figure shows the results of anti-HA ChIP-qPCR analysis in MCF7 cells exhibiting ectopic expression of LCOR in a form fused with HA. Conditions included LCOR (LCOR wild-type); LSKAA (LCOR with two amino acid changes in the nuclear receptor domain); and delta-HTH (ΔHTH; LCOR without the HTH domain). n=3 independent biological replicates; data represent mean ± SEM. [Figure 11] This figure shows flow cytometry analysis of anti-pan-HLA-ABC in MCF7 cells exhibiting ectopic expression of LCOR and LSKAA. n=3 independent biological replicates; data represent relative mean fluorescence intensity (MFI) ± SEM. [Figure 12] This is a diagram illustrating LCOR fragments. It is a schematic diagram of the LCOR protein structure and domains, including different LCOR fragments constructed with stepwise deletions of the indicated protein domains. Delta symbols represent deletions of the indicated domains. These fragments are designed for ectopic protein expression using the pLEX-MCS plasmid backbone. [Figure 13] This figure shows the functional testing of LCOR fragments in MHC-I regulation. (A) Flow cytometry analysis of MDA-MB-231 cells transduced to overexpress the shown fragment compared to a control condition (pLEX; empty vector). Anti-pan-HLA-ABC-PECy5 measures pan-HLA-ABC expression on the cell surface of these cells. (B) Bar plot of relative mean fluorescence intensity (MFI) of flow cytometry analysis of the shown LCOR fragment. [Figure 14] This figure shows the analysis of antigen presentation and processing mechanisms (APM) in MDA-MB-231(NR-) and MCF7(NR+) cell lines treated with various LCOR fragments. A: MCF7(NR+) overexpressing different LCOR protein fragments. Normalized to empty vector expression. Data represent mean values. B: Schematic diagrams of LCOR WT and fragments, as well as the main interaction regions described in the experiment. [Figure 15] This is a diagram showing the predicted structures of LCOR wt, LCOR-LSKAA, and LCOR-dNR. [Figure 16] A) This figure shows cellular immunohumanization by transplanting ER+ MCF7 breast cancer cells and human peripheral blood mononuclear cells (PBMCs) into immunodeficient mice (NSGs). The plots show in vivo tumor growth under the indicated conditions. B) This figure shows tumor-infiltrating lymphocytes (TILs) under the indicated conditions. [Figure 17] This figure shows a co-culture of chumoroids derived from an ER+BC tumor sample (patient BC-201T), transduced from human peripheral blood mononuclear cells (PBMCs) and LSKAA counter-control. [Modes for carrying out the invention]

[0007] The present invention provides novel and improved cancer therapies based on isolated or synthesized mutants or fragments of ligand-gated corepressors (LCORs), or based on polynucleotide sequences encoding such mutants or fragments of ligand-gated corepressors (LCORs), wherein the mutants or fragments of ligand-gated corepressors (LCORs) are characterized by containing an HTH domain, and further characterized by the absence or inactivation of a nuclear receptor-binding domain.

[0008] Please note that, as used herein, LCoR may also be referred to as LCOR.

[0009] In the context of this invention, "ligand-dependent corepressor (LCOR)" is understood as the wild-type form of LCOR. Functionally, it is a transcription factor that induces the transcriptional expression of antigen-presenting mechanism (APM) genes, particularly when it does not interact with nuclear receptors. Its function is It can be lost through interaction with nuclear receptors (e.g., estrogen receptors in particular). The complete LCOR sequence is shown in Figure 12 of the present invention. The complete protein and DNA sequences of the wild-type LCOR form are shown in Sequence 1 and Sequence 2 below, respectively.

[0010] Protein sequence (SEQ ID NO: 1) MQRMIQQFAAEYTSKNSSTQDPSQPNSTKNQSLPKASPVTTSPTAATTQNPVLSKLLMADQDSPLDLTVRKSQSEPSEQDGVLDLSTKKSPCAGSTSLSHSPGCSSTQ GNGRPGRPSQYRPDGLRSGDGVPPRSLQDGTREGFGHSTSLKVPLARSLQISEELLSRNQLSTAASLGPSGLQNHGQHLILSREASWAKPHYEFNLSRMKFRGNGALS NISDLPFLAENSAFPKMALQAKQDGKKDVSHSSPVDLKIPQVRGMDLSWESRTGDQYSYSSLVMGSQTESALSKKLRAILPKQSRKSMLDAGPDSWGSDAEQSTSGQP YPTSDQEGDPGSKQPRKKRGRYRQYNSEILEEAISVVMSGKMSVSKAQSIYGIPHSTLEYKVKERLGTLKNPPKKKMKLMRSEGPDVSVKIELDPQGEAAQSANESKNE

[0011] cDNA sequence (SEQ ID NO: 2)

[0012] In the context of the present invention, “ligand-dependent corepressor (LCOR) variant or fragment” is understood as a more preferred and improved fragment or form of a ligand-dependent corepressor (LCOR) that can induce transcriptional expression of antigen-presenting mechanism (APM) genes, as measured by flow cytometry analysis of MDA-MB-231 cells transduced, transformed, or transfected to overexpress a specific LCOR fragment or variant, by using an antibody to measure pan-HLA-ABC expression on the cell surface of these transduced cells by fluorescence intensity (MFI) (e.g., by using an antibody such as anti-pan-HLA-ABC-PECy5 (clone W6 / 32) (see Perez-Nunez et al. Nat Cancer 2022)). Such pan-HLA-ABC expression by fluorescence intensity (MFI) on the cell surface of these transduced cells must be at least 1.5-fold, 2-fold, 3-fold, 4-fold, or at least 10-fold change or increase compared to control cells, MDA-MB-231 cells, transduced using an empty vector. It should be noted that in this invention, all variants and fragments of the present invention avoid interaction with nuclear receptors.

[0013] In the context of the present invention, the "nuclear receptor-binding domain" is understood as the domain in which the LCOR interacts with nuclear receptors such as estrogen receptors.

[0014] Surprisingly, the inventors of this invention have found that, as long as at least the HTH domain is retained, the transcriptional expression of the antigen presentation mechanism (APM) gene can be induced, ligand-dependent coliform We discovered several mutants or fragments of the presser (LCOR). Furthermore, some of these specific mutants or fragments significantly improved the anticancer activity of LCOR. Notably, in this regard, see Figure 8, which shows that LSKAA alone (without the need to combine it with atezolizumab) significantly reduced cell viability compared to the control, and even significantly reduced cell viability compared to the combination of LCOR and atezolizumab.

[0015] Accordingly, a first aspect of the present invention refers to an isolated or synthesized mutant or fragment of a ligand-dependent corepressor (LCOR), or a polynucleotide sequence encoding such mutant or fragment of a ligand-dependent corepressor (LCOR), wherein the mutant or fragment of the ligand-dependent corepressor (LCOR) is characterized by containing an HTH domain, and further characterized by the absence or inactivation of a nuclear receptor-binding domain.

[0016] As stated above, it should be noted that "ligand-dependent corepressor (LCOR) variants or fragments" are understood as more preferred and improved fragments or forms of ligand-dependent corepressors (LCORs) that can induce transcriptional expression of antigen-presenting mechanism (APM) genes, as measured by flow cytometry analysis of MDA-MB-231 cells that have been transduced, transformed, or transfected to overexpress a specific LCOR fragment or variant, by using an antibody to measure pan-HLA-ABC expression on the cell surface by fluorescence intensity (MFI) (for example, by using an antibody such as anti-pan-HLA-ABC-PECy5 (clone W6 / 32) (see Perez-Nunez et al. Nat Cancer 2022)). The expression of pan-HLA-ABC, as measured by the fluorescence intensity (MFI) on the cell surface of these transduced cells, must be at least 1.5-fold, 2-fold, 3-fold, 4-fold, or at least 10-fold compared to MDA-MB-231 cells, which are control cells transduced using an empty vector. It should be noted that in this invention, all variants and fragments of the present invention avoid interaction with nuclear receptors.

[0017] Furthermore, it should be noted that the term “polynucleotide sequence” as used in the first embodiment of the present invention described above refers to any oligonucleotide or polynucleotide molecule, including DNA and / or RNA oligonucleotides or polynucleotide molecules, and “oligonucleotide or polynucleotide analogs.” An “oligonucleotide or polynucleotide analog” is a molecule derived from a DNA and / or RNA oligonucleotide or polynucleotide molecule, wherein at least one of the nucleotide units constituting the DNA and / or RNA oligonucleotide or polynucleotide molecule incorporates several chemical modifications, for example, a phosphate group, a pentose, or a nitrogen-containing base. With respect to chemical modifications that may be included in oligonucleotide or polynucleotide analogs, this term applies, in particular, to one or more of the usual modifications known to those skilled in the art of molecular biology, from the perspective of basic research, especially the investigation of the therapeutic applications of these molecules.

[0018] In a preferred embodiment of the first aspect of the present invention, the HTH domain is characterized by comprising or consisting solely of the amino acid sequence VSKAQSIYGIPHSTLEYKVKE (SEQ ID NO: 3). Preferably, the HTH domain is characterized by comprising, or consisting solely of, or substantially consisting solely of, the amino acid sequence VSKAQSIYGIPHSTLEYKVKE, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to VSKAQSIYGIPHSTLEYKVKE (SEQ ID NO: 3).

[0019] In another preferred embodiment of the first aspect of the present invention, a ligand-dependent corepressor A variant or fragment of (LCOR) can induce transcriptional expression of an antigen presentation mechanism (APM) gene and is characterized by containing the amino acid sequence VSKAQSIYGIPHSTLEYKVKE (SEQ ID NO: 3), or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to VSKAQSIYGIPHSTLEYKVKE (SEQ ID NO: 3), and the NLS motif of the amino acid sequence PRKKRGR (SEQ ID NO: 4), or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO: 4. Preferably, the ligand-dependent corepressor (LCOR) variant or fragment can induce transcriptional expression of antigen-presenting mechanism (APM) genes and is characterized by containing, consisting solely of, or substantially consisting solely of, a sequence identical to, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% of the amino acid sequence SEQ ID NO: 5. Preferably, the ligand-dependent corepressor (LCOR) variant or fragment can induce transcriptional expression of antigen-presenting mechanism (APM) genes and is characterized by containing, consisting solely of, or substantially consisting solely of, a sequence identical to, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% of the amino acid sequence SEQ ID NO: 7.

[0020] In the context of this invention, the “NLS motif” is understood as an amino acid sequence that “tags” a protein for translocation into the cell nucleus.

[0021] >LCOR_HTH Protein sequence (SEQ ID NO: 5) TIFF2026510428000001.tif10170

[0022] cDNA sequence (SEQ ID NO: 6) ATGCCTCGGAAGAAAAGAGGGCGTTACAGACAGTACAACAGTGAGATACTGGAGGAAGCAATCTCAGTGGTTATGAGTGGAAAAATGAGTGTTTCCAAAGCTCAGAGTATTTATGGGATTCCCCACAGTACACTGGAGTACAAA GTAAAGGAGAGGCTGGGCACTTTGAAAAACCCTCCAAAGAAAAAGATGAAATTAATGAGGTCGGAGGGGCCAGATGTTTCTGTAAAGATTGAATTAGATCCCAGGGAGAGGCAGCACAAAGTGCAAATGAATCAAAAAACGAG

[0023] >LCOR_HTH-IDP Protein sequence (SEQ ID NO: 7) TIFF2026510428000002.tif10170

[0024] cDNA sequence (SEQ ID NO: 8) ATGCAAAGTAGAAAAAGCATGTTAGATGCTGGACCCGATTCTTGGGGCTCAGATGCTGAGCAGTCTACCTCTGGACAGCCATATCCCACATCGGATCAAGAAGGAGACCCTGGCTCCAAGCAGCCTCGGAAGAAAAGAGGGCGTTACAGACAGTACAACAGTGAGATACTGGAGGAAGCAATCTCAGTGGTTATGAGTGGAAAA ATGAGTGTTTCCAAAGCTCAGAGTATTTATGGGATTCCCCACAGTACACTGGAGTACAAAGTAAAGGAGAGGCTGGGCACTTTGAAAAACCCTCCAAAGAAAAAGATGAAATTAATGAGGTCGGAGGGGCCAGATGTTTCTGTAAAGATTGAATTAGATCCCCAGGAGAGGCAGCACAAAGTGCAAATGAATCAAAAAACGAG

[0025] The term "sequence identity" refers to the percentage value obtained when two sequences are compared using a pairwise sequence alignment tool. In this specification, sequence identity is obtained using the global alignment tool "EMBOSS Needle" with default settings (Rice et al., 2000. Trends Genet. 16(6):276-7, Li et al.). ., 2015. Nucleic Acids Res. 43(W1):W580-4). The global alignment tool is available at https: / / www.ebi.ac.uk / Tools / psa / .

[0026] In another preferred embodiment of the first aspect of the present invention, the ligand-dependent corepressor (LCOR) variant or fragment is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence VSKAQSIYGIPHSTLEYKVKE (SEQ ID NO: 3), or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% identical to the amino acid sequence PRKKRGR (SEQ ID NO: 4), or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% identical to SEQ ID NO: 4. The present invention is characterized by an amino acid sequence comprising at least one NLS motif of a sequence that is %, 98%, or 100% identical, and an HDAC motif of a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence NLSRMKFRGNGALSNISDLPFLAENSAFPKMALQAKQDGKKDVSHSSPVDLKIPQVRGMDLSWESRTGDQYSYSSLVMGSQTESALSKKLRAILPKQSRKSMLDAGPDSWGSDAEQS (SEQ ID NO: 9), or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO: 9. Preferably, the ligand-dependent corepressor (LCOR) variant or fragment is characterized by containing, consisting solely of, or substantially consisting solely of, the amino acid sequence of SEQ ID NO: 10, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO: 10. Preferably, the ligand-dependent corepressor (LCOR) variant or fragment is characterized by containing, consisting solely of, or substantially consisting solely of, the amino acid sequence of SEQ ID NO: 12, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO: 12.

[0027] >LCOR_HDAC Protein sequence (SEQ ID NO: 10) NLSRMKFRGNGALSNISDLPFLAENSAFPKMALQAKQDGKKDVSHSSPVDLKIPQVRGMDLSWESRTGDQYSYSSLVMGSQTESALSKKLRAILPKQSRKSMLDAGPDSWGSDAEQSTSGQPYPTSDQEGDPGSKQPRKKRGRYRQYNSEILEEAISVVMSGKMSVSKAQSIYGIPHSTLEYKVKERLGTLKNPPKKKMKLMRSEGPDVSVKIELDPQGEAAQSANESKNE

[0028] cDNA sequence (SEQ ID NO: 11) ATGAACCTCAGCCGTATGAAGTTCAGGGGAAATGGTGCACTCAGCAACATCAGTGACCTTCCTTTTCTTGCAGAAAACTCTGCCTTTCCAAAAATGGCACTTCAAGCAAAACAAGATGGAAAAAAGGATGTGAGCCATTCATCTCCTGTAGATTTAAAGATACCACAAGTTCGAGGAATGGATCTTTCTTGGGAGTCTCGCACTGGTGATCAGTACAGCTATAGCTCTTTGGTAATGGGTTCACAAACGGAGAGCGCGCTTAGTAAAAAATTAAGGGCTATTCTTCCAAAACAAAGTAGAAAAAGCATGTTAGATGCTGGACCCGATTCTTGGGGCTCAGATGCTGAGCAGTCTACCTCTGGACAGCCATATCCCACATCGGATCAAGAAGGAGACCCTGGCTCCAAGCAGCCTCGGAAGAAAAGAGGGCGTTACAGACAGTACAACAGTGAGATACTGGAGGAAGCAATCTCAGTGGTTATGAGTGGAAAAATGAGTGTTTCCAAAGCTCAGAGTATTTATGGGATTCCCCACAGTACACTGGAGTACAAAGTAAAGGAGAGGCTGGGCACTTTGAAAAACCCTCCAAAGAAAAAGATGAAATTAATGAGGTCGGAGGGGCCAGATGTTTCTGTAAAGATTGAATTAGATCCCCAGGGAGAGGCAGCACAAAGTGCAAATGAATCAAAAAACGAG

[0029] >LCOR_ΔNR / ΔCTBP Protein sequence (SEQ ID NO: 12) KSPCAGSTSLSHSPGCSSTQGNGRPGRPSQYRPDGLRSGDGVPPRSLQDGTREGFGHSTSLKVPLARSLQISEELLSRNQLSTAASLGPSGLQNHGQHLILSREASWAKPHYEFNLSRMKFRGNGALSNISDLPFLAENSAFPKMALQAKQDGKKDVSHSSPVDLKIPQVRG MDLSWESRTGDQYSYSSLVMGSQTESALSKKLRAILPKQSRKSMLDAGPDSWGSDAEQSTSGQPYPTSDQEGDPGSKQPRKKRGRYRQYNSEILEEAISVVMSGKMSVSKAQSIYGIPHSTLEYKVKERLGTLKNPPKKKMKLMRSEGPDVSVKIELDPQGEAAQSANESKNE

[0030] cDNA sequence (SEQ ID NO: 13)

[0031] In another preferred embodiment of the first aspect of the present invention, the ligand-dependent corepressor (LCOR) variant or fragment is further characterized by comprising either a CTBP1 binding site or / or a CTBP2 binding site. Preferably, the ligand-dependent corepressor (LCOR) variant or fragment is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence VSKAQSIYGIPHSTLEYKVKE (SEQ ID NO: 3), or a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence PRKKRGR (SEQ ID NO: 4), or a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, or 91% identical to the amino acid sequence SEQ ID NO: 9, The present invention is characterized by an amino acid sequence containing an HDAC motif having a sequence that is 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical, and a CTBP1 binding site and / or a CTBP2 binding site, wherein the CTBP1 binding site is a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence PLDLTVR (SEQ ID NO: 14) or SEQ ID NO: 14, and the CTBP2 binding site is a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence VLDLSTK (SEQ ID NO: 15) or SEQ ID NO: 15. Preferably, the ligand-dependent corepressor (LCOR) variant or fragment is characterized by containing, consisting solely of, or substantially consisting solely of, the amino acid sequence of SEQ ID NO: 16, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO: 16.

[0032] In the context of the present invention, the "CTBP1 binding site" is understood as a protein domain that interacts with CTBP1 (C-terminal binding protein 1).

[0033] In the context of the present invention, the "CTBP2 binding site" is understood as a protein domain that interacts with CTBP2 (C-terminal binding protein 2).

[0034] >LCOR_ΔNR Protein sequence (SEQ ID NO: 16) PLDLTVRKSQSEPSEQDGVLDLSTKKSPCAGSTSLSHSPGCSSTQGNGRPGRPSQYRPDGLRSGDGVPPRSLQDGTREGF GHSTSLKVPLARSLQISEELLSRNQLSTAASLGPSGLQNHGQHLILSREASWAKPHYEFNLSRMKFRGNGALSNISDLPFLAENSAFPKMALQAKQDGKKDVSHSSPVDLKIPQVRGMDLSWESRTGDQYSYSSLVMGSQTESAL SKKLRAILPKQSRKSMLDAGPDSWGSDAEQSTSGQPYPTSDQEGDPGSKQPRKKRGRYRQYNSEILEEAISVVMSGKMSVSKAQSIYGIPHSTLEYKVKERLGTLKNPPKKKMKLMRSEGPDVSVKIELDPQGEAAQSANESKNE

[0035] cDNA sequence (SEQ ID NO: 17)

[0036] In another preferred embodiment of the first aspect of the present invention, a ligand-dependent corepressor (LCOR) variant or fragment is characterized by containing, or consisting solely of, an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO: 18.

[0037] >LCOR_LSKAA Protein sequence (SEQ ID NO: 18) MQRMIQQFAAEYTSKNSSTQDPSQPNSTKNQSLPKASPVTTSPTAATTQNPVLSKAAMADQDSPLDLTVRKSQSEPSEQDGVLDLSTKKSPCAGSTSLSHSPGCSSTQ GNGRPGRPSQYRPDGLRSGDGVPPRSLQDGTREGFGHSTSLKVPLARSLQISEELLSRNQLSTAASLGPSGLQNHGQHLILSREASWAKPHYEFNLSRMKFRGNGALS NISDLPFLAENSAFPKMALQAKQDGKKDVSHSSPVDLKIPQVRGMDLSWESRTGDQYSYSSLVMGSQTESALSKKLRAILPKQSRKSMLDAGPDSWGSDAEQSTSGQP YPTSDQEGDPGSKQPRKKRGRYRQYNSEILEEAISVVMSGKMSVSKAQSIYGIPHSTLEYKVKERLGTLKNPPKKKMKLMRSEGPDVSVKIELDPQGEAAQSANESKNE

[0038] cDNA sequence (SEQ ID NO: 19) ACAGTACAACAGTGAGATACTGGAGGAAGCAATCTCAGTGGTTATGAGTGGAAAAATGAGTGTTTCCAAAGCTCAGAGTATTTATGGGATTCCCCACAGTACACTGGAGTACAAAGTAAAGGAGAGGCT GGGCACTTTGAAAAACCCTCCAAAGAAAAAGATGAAATTAATGAGGTCGGAGGGGCCAGATGTTTCTGTAAAGATTGAATTAGATCCCCAGGGAGAGGCAGCACAAAGTGCAAATGAATCAAAAAACGAG

[0039] In another preferred embodiment of the first aspect of the present invention, a ligand-dependent corepressor (LCOR) variant or fragment is characterized by comprising, or consisting solely of, an amino acid sequence identical to, amino acid sequence number 18 (LSKAA variant), or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to sequence number 18.

[0040] In another preferred embodiment of the first aspect of the present invention, the ligand-dependent corepressor (LCOR) variant or fragment consists solely of amino acid sequence number 18 (LSKAA variant).

[0041] In another preferred embodiment of the first aspect of the present invention or any of its preferred embodiments, the polynucleotide is a polyribonucleotide sequence comprising a DNA sequence, for example, a cDNA (complementary DNA) sequence, or a stabilized mRNA or mRNA-derived sequence, wherein the DNA sequence or polyribonucleotide sequence can be efficiently translated into a variant or fragment sequence of any of the first aspects of the present invention or any of its preferred embodiments.

[0042] A second aspect of the present invention refers to an expression vector, polynucleotide DNA / RNA (e.g., naked mRNA), or plasmid, wherein the polynucleotide of the first aspect of the present invention is located within, inserted into, or contained within the expression vector, polynucleotide DNA / RNA (e.g., naked mRNA), or plasmid, wherein the expression vector, polynucleotide DNA / RNA (e.g., naked mRNA), or plasmid is selected for or to enable its ability to express (transcribe and translate) the (LCOR) variant or fragment thereof of the first aspect of the present invention in mammalian cells, preferably human cells, more preferably human cancer.

[0043] As used herein, an expression vector is designed to enable the expression (transcription and translation) of an inserted section of DNA / RNA (a polynucleotide in the first aspect of the present invention). An expression vector typically has a promoter and a transcription terminator.

[0044] A preferred embodiment of the second aspect of the present invention refers to an expression vector, polynucleotide DNA / RNA (e.g., naked mRNA), or plasmid, or a mixture of any combination thereof, wherein at least a portion of the expression vector, polynucleotide DNA / RNA (e.g., naked mRNA), or plasmid, or any combination thereof, is selected for its ability to express a (LCOR) variant or fragment of the first aspect of the present invention in mammalian cells, preferably human cells, more preferably human cancer, and another portion of the expression vector, polynucleotide DNA / RNA (e.g., naked mRNA), or plasmid, or any combination thereof, is selected for its ability to induce the presentation of one or more nascent antigens in mammalian cells, preferably human cells, more preferably human cancer.

[0045] In a third aspect of the present invention, the present invention relates to isolated or synthesized mutants or fragments of ligand-dependent corepressors (LCORs) according to the first aspect of the present invention, or polynucleotides encoding them, or to expression vectors, polynucleotide DNA / RNA (e.g., naked mRNA), or plasmids according to the second aspect of the present invention, or any thereof. The present invention provides compositions that enable in vitro or in vivo delivery of a combination of cells or tissues, such as mammalian cells, preferably human cells, and more preferably human cancer cells. Such compositions are also referred to herein as “carriers” or “vehicles.”

[0046] In a preferred embodiment of a third aspect of the present invention, the composition enabling the delivery of the expression vector, polynucleotide DNA / RNA (e.g., naked mRNA), or plasmid described above is a nano or microemulsion or nano or microsystem, exosome, lipid nanoparticles, polymer nanoparticles, nanoparticles, natural or artificial lipoprotein particles, cationic lipids, protein nucleic acid complexes, liposomes, visomes, or polymers. In embodiments of the present invention, the vector is a viral vector, including lentiviruses and AAV (adeno-associated virus).

[0047] A fourth aspect of the present invention refers to any of the compositions described in the first to third aspects of the present invention, used for therapeutic purposes or as a pharmacologic agent, particularly an LCOR variant or fragment or a polynucleotide encoding it according to the first aspect of the present invention, an expression vector, polynucleotide DNA / RNA (e.g., naked mRNA), a plasmid, or any combination thereof according to the second aspect of the present invention, or a composition enabling the delivery of such LCOR variant or fragment, or an expression vector, polynucleotide DNA / RNA (e.g., naked mRNA), or plasmid according to the third aspect of the present invention. Preferably used in immunotherapy, particularly in cancer immunotherapy, and more specifically, in the treatment of cancer, such as breast cancer or triple-negative breast cancer (TNBC).

[0048] A fifth aspect of the present invention refers to any of the compositions described in the first to third aspects of the present invention, used in a method for treating cancer, preferably in a method for cancer immunotherapy, particularly an LCOR variant or fragment or a polynucleotide encoding it according to the first aspect of the present invention, an expression vector, polynucleotide DNA / RNA (e.g., naked mRNA), plasmid, or any combination thereof according to the second aspect of the present invention, or a composition that enables the delivery of the LCOR variant or fragment, or an expression vector, polynucleotide DNA / RNA (e.g., naked mRNA), or plasmid according to the third aspect of the present invention. In a preferred embodiment of the fifth aspect of the present invention, the cancer is characterized by comprising or consisting solely of cancer cells that are androgen or estrogen or progesterone receptor positive (AR+ or ER+ or PGR+), or progesterone receptor positive, or any other nuclear receptor (NR) positive. Preferably, the cancer is characterized by comprising or consisting solely of cancer cells that are androgen or estrogen receptor positive (AR+ or ER+). More preferably, the cancer is characterized by comprising or consisting solely of estrogen receptor-positive (ER+) cancer cells.

[0049] As used herein, the terms "androgen or estrogen receptor positive (AR+ or ER+), or progesterone receptor (PGR+), or any other nuclear receptor (NR) positive" are understood to mean nuclear receptor proteins that can interact with the wild-type form of LCOR.

[0050] In another preferred embodiment of the fifth aspect of the present invention, cancer is chondrosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma / osteosarcoma of bone, osteosarcoma, rhabdomyosarcoma, cardiac cancer, astrocytoma, brainstem glioma, pilocytic astrocytoma, ependymoma, undifferentiated neuroectodermal tumor, cerebellar astrocytoma, cerebral astrocytoma, glioma, medulloblastoma, neuroblastoma, oligodendroglioma, pineal astrocytoma, pituitary adenoma, Visual pathway hypothalamic glioma, breast cancer, invasive lobular carcinoma, tubular carcinoma, invasive cribriform carcinoma, medullary carcinoma, male breast cancer, phyllodes tumor, inflammatory breast cancer, adrenocortical carcinoma, islet cell carcinoma (pancreatic endocrine part), multiple endocrine neoplasia syndrome, parathyroid carcinoma, pheochromocytoma, thyroid cancer, Merkel cell carcinoma, uveal melanoma, retinoblastoma, anal cancer, appendiceal cancer, bile duct cancer, carcinoid tumor, colorectal cancer, extrahepatic bile duct cancer, gallbladder cancer, Gastric (stomach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, hepatocellular carcinoma, pancreatic cancer, islet cell carcinoma, rectal cancer, bladder cancer, cervical cancer, endometrial cancer, extragonadal germ cell tumors, ovarian cancer, ovarian epithelial carcinoma (surface epithelial-stromal tumors), ovarian germ cell tumors, penile cancer, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, prostate cancer, testicular cancer, gestational trophoblastic neoplasm, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms' tumor, esophageal cancer, head and neck cancer, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, paranasal sinus cancer and nasal cavity cancer, pharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, basal cell carcinoma, squamous cell carcinoma, skin adnexal tumors (e.g., sebaceous gland carcinoma), melanoma, skin The cancer is selected from primary sarcomas (e.g., dermatofibrosarcoma protuberans), primary cutaneous lymphomas (e.g., mycosis fungoides), bronchial adenomas / carcinoids, small cell lung cancer, mesothelioma, non-small cell lung cancer, pleuroblastoma, laryngeal cancer, thymoma and thymic carcinoma, Kaposi's sarcoma, epithelioid hemangioendothelioma (EHE), fibrinogenic small round cell tumors, and liposarcoma, and preferably the cancer is selected from lung cancer, melanoma, kidney cancer, bladder cancer, prostate cancer, mesothelioma, glioblastoma, and breast cancer, and more preferably the cancer is selected from melanoma, bladder cancer, and breast cancer, preferably ER+ breast cancer or triple-negative breast cancer (TNBC).

[0051] In another preferred embodiment of a fifth aspect of the present invention, the treatment method is performed before, simultaneously with, or after cancer treatment with an anticancer agent. Preferably, the anticancer agent is selected from, but is not limited to, adoptive cell transfer or an immune checkpoint inhibitor (ICI). That is, in this embodiment, any of the compositions described in the first to third aspects of the present invention may be administered before, simultaneously with, or sequentially with the anticancer agent. Such combination therapy comprising any of the compositions described in the first to third aspects of the present invention and an anticancer agent, such as adoptive cell transfer or an immune checkpoint inhibitor (ICI), can be achieved by administering any of the compositions described in the first to third aspects of the present invention and the anticancer agent simultaneously. Such combination therapy can be achieved by administering a single pharmaceutical composition containing both agents, or by administering two different pharmaceutical compositions simultaneously, one of which comprises any of the compositions described in the first to third aspects of the present invention and the other comprises an anticancer agent. Alternatively, treatment with any of the compositions described in the first to third aspects of the present invention may precede or follow treatment with an anticancer agent at intervals ranging from several minutes to several weeks.

[0052] In a preferred embodiment, the composition of the fifth aspect of the present invention is administered before or after surgery.

[0053] As used herein, “anticancer agent” is understood to mean a therapeutic agent used to inhibit, reduce, or eliminate cancer cells.

[0054] Examples of immune checkpoint inhibitors include drugs that inhibit, block, or reduce CTLA-4, PD-1, PD-L1, Lag-3, Tim-3, TIGIT, etc. Suitable anti-CTLA-4 therapeutic agents used in the present invention include, but are not limited to, anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA-4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, ipilimumab, tremelimumab, anti-CD28 antibodies, anti-CTLA-4 adnectin, anti-CTLA-4 domain antibodies, anti-CTLA-4 single-chain fragments, anti-CTLA-4 heavy-chain fragments, anti-CTLA-4 light-chain fragments, and CTLA-4 inhibitors that stimulate the costimulatory pathway. Suitable anti-PD-1 and anti-PD-L1 therapeutic agents used in the present invention include, but are not limited to, anti-PD-1 and anti-PD-L1 antibodies, human anti-PD-1 and anti-PD-L1 and anti-PD-L1 antibodies, mouse anti-PD-1 and anti-PD-L1 antibodies, mammalian anti-PD-1 and anti-PD-L1 antibodies, humanized anti-PD-1 and anti-PD-L1 antibodies, monoclonal anti-PD-1 and anti-PD-L1 antibodies, polyclonal anti-PD-1 and anti-PD-L1 antibodies, and chimeric anti-PD-1 and anti-PD-L1 antibodies. In certain embodiments, anti-PD-1 therapeutic agents include nivolumab, pembrolizumab, and pidi. Examples include lizumab, MEDI0680, and combinations thereof. In other specific embodiments, examples of anti-PD-L1 therapeutic agents include atezolizumab, BMS-936559, MEDI4736, MSB0010718C, and combinations thereof. Preferred anti-Lag-3 therapeutic agents used in the present invention include, but are not limited to, BMS-986016 and TSR-033. Preferred anti-Tim-3 therapeutic agents used in the present invention include, but are not limited to, TSR-022 (covolimab). Preferred anti-TIGIT therapeutic agents used in the present invention include, but are not limited to, BMS-986207.

[0055] The following examples are merely illustrative of the present invention and do not limit it. [Examples]

[0056] Materials and methods Ethical regulations This study complies with all ethical regulations. Clinical patient samples were reviewed by the Clinical Research Ethics Committee (Mar Park of Health), the Vall d'Hebron Hospital Institutional Review Board, and INCLIVA's Ethics Board. The study has been approved by the committee. All individuals submitted informed consent before inclusion. All animal research procedures described in this study have been approved by the Animal Research Ethics Committee of Barcelona Biomedical Research Park. Approved by the Generalitat de Catalunya (Catalan government). In all experimental procedures, the tumor was 1500 mm. 3 Euthanasia was performed when the animal reached a certain volume or when its health deteriorated.

[0057] Animal testing This study used mouse strains Balb / c, C57BL / 6J, C57BL / 6-Tg(TcraTcrb)1100Mjb / J(OT-1), Ptprca(TcrbTcra)Ln1Bdb H2d / J Just Enhanced GFP(JEDI), and NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ(NSG). Tumor cells were orthotopically injected into MFPs using a 1:1 PBS:Matrigel (Corning) mixture. To evaluate TIC, LDA cells were orthotopically transplanted into severely immunodeficient NSG female mice. After two weeks, tumor development was assessed by palpation, and the TIC frequency was calculated using extreme limiting dilution analysis (ELDA) software.16 For tumor comparison between immunocompetent and immunodeficient strains, 5000 AT3 or 4TO7 cells were transplanted into 10 mammary MFPs of NSG and Balb / c or C57BL / 6J mice. Tumor volume was measured twice weekly using a digital caliper, and calculations were applied (π × length × width 2 / 6). In the ICB assay, mice were treated with either anti-human / mouse anti-PD-L1 (atezolizumab, clone SP142, Tecentriq) or anti-mouse anti-PD-L1 (BioXCell, clone 10F.9G2, catalog number: BE0101) as the vehicle control. Tumor size: 0.5 x 0.5 cm 2 Treatment was initiated when the tumor reached a certain level. A 10 mg / kg dosing regimen was applied every 3 or 7 days (as shown in separate experiments). CD4 and CD8 depletion was diagnosed using anti-CD4 and anti-CD8 neutralizing antibodies, respectively, when the tumor was 0.5 × 0.5 cm. 2 Once the target was reached, 400 μg was administered per mouse, followed by 250 μg once weekly. In lung metastasis experiments, 50,000 transduced control cells and 150,000 Lcor-overexpressing 4TO7 cells were TV-injected into Balb / c mice, achieving similar metastatic growth between conditions. One week later, the metastatic sites were treated weekly with 10 mg / kg of anti-PD-L1. Metastatic lesions were monitored weekly by bioluminescent photon flux imaging (BLI), and data were collected using Live Image v.4.3.1 of the Perkin Elmer Living Image system. In the humanized xenograft model, 15,000 MDA-MB-231 cells were orthotopically transplanted into NSG mice. The tumor size was 0.3 × 0.3 cm. 2 Once it reaches that point, the animals will receive 10 units derived from 63 healthy donors. 000000 peripheral blood mononuclear cells (PBMCs) were injected intraperitoneally. The tumor size was 0.5 × 0.5 cm. 2Once reached, the animals were treated every three days with either vehicle or 10 mg / kg of human / mouse cross-reactive anti-PD-L1.

[0058] Paraffin-embedded tissue samples and PDO Paraffin-embedded tissue samples were collected before and after treatment from a total of four patients with initial TNBC. Two patients were treated with the combination of combination chemotherapy (Abraxane + carboplatin) and atezolizumab (NeoTRIP trial), while the other two were treated with the combination of TAK649 and anti-PD1 nivolumab (NCT02834247 trial). The samples were obtained from the Hospital Clinic of Valencia. The hormone receptor status was evaluated by IHC as (ER - and PR- were defined as positive staining nuclei less than 1%), and HER2 was evaluated by IHC and fluorescence in situ hybridization.

[0059] Patient-derived tumor fragments (about 1 cm 3 ~5 cm 3 ) were collected by surgical resection at the Hospital del Mar (Barcelona) from patients with TNBC who had given prior informed consent. The tumor fragments were mechanically and enzymatically digested for 2 hours at 37°C in mammary epithelial cell growth medium 16 supplemented with enzymes, and then incubated briefly with 5 mg / ml of dispase (Merck, catalog number : D4693), 0.1 mg / ml of DNase I (Merck, catalog number: D5025 : -150), and 0.25% trypsin (Cultek, catalog number: 25300-062). The digested tissue suspension was filtered through a 100 μm filter, and single cells were cultured and grown in vitro as previously described.

[0060] Cell lines, culture conditions, and treatments All cell lines used in this study (breast cancer cell lines (human MDA-MB-231, MCF7, and mouse Py8119, 4TO7, and AT3)) were provided by Y. Kang of Princeton University and cultured according to the American Type Culture Collection (ATCC). HMLE cells were provided by R. Weinberg of MIT. HEK293T cells were cultured according to the ATC. The cells were obtained from C. The cells were routinely tested for mycoplasma and all were negative. In this study, anti-human / mouse anti-PD-L1 (atezolizumab, clone SP142, Tecentriq) was used in vitro at a concentration of 10 μg / ml for 72 hours. Recombinant mice or human IFNγ (R&D systems, human: catalog number: 285-IF-100; ma) were used. (Us: Catalog number: 485-MI-100) was used at 10 ng / ml for 24 hours, and the JAK1-JAK2 inhibitor ruxolitinib (LC laboratories, catalog number: R-6688) was used. ) was used at 1 μM for 24 hours, BX-795 (TBK1i) (Merck, catalog number: S ML0694) was used at 0.5 nM for 24 hours.

[0061] CTL assay Splenocytes were collected from OT-I and JEDI mouse models for mouse cell co-culture assays. The spleens were mechanically minced, and single cells were incubated in ACK buffer (Fisher, catalog number: A1049201). The single-cell suspensions were filtered through a 70 μm filter, and OT-1 and JEDI cells were activated overnight in mouse splenocyte medium with 2 μg / ml of SIINFEKL peptide (Merck, catalog number: S7951) or HYLSTQSAL peptide (Proimmune, catalog number: F198-2A-E), respectively. CD8 + T cells were negatively selected using the CD8a+ T cell isolation kit (Milteny, catalog number: 130-104-075). The cells were purified and then co-cultured with tumor cells for 72 hours with or without 10 μg / ml of anti-human / mouse PD-L1 (reference 65) (atezolizumab, clone SP142, Tecentriq). In a T-cell killing assay using β2m knockdown, the cell line was treated with lipofectamine 3000 (Life Technologies, catalog number: L3000015). Use a 100ρM siRNA-negative control (Life Technologies, catalog number: AM4). 613) or 100ρM Silencer Pre-designed siRNA (Life Technologies, catalog number: 160820) against mouse β2m Translocation was performed using [a specific method]. In the human co-culture assay, PBMCs were isolated by stepwise centrifugation of blood samples from healthy adult donors obtained from the Tissue Bank of Catalonia and co-cultured with tumor cells in co-culture medium. After 72 hours, cell viability or enrichment was measured by crystal violet method or flow cytometry, respectively.

[0062] EV isolation and in vitro / in vivo delivery assays HEK293T cells were cultured in DMEM with 10% reduced extracellular vesicle (EV) fetal bovine serum (FBS) (Hyclone), and pLEX-H was treated with lipofectamine 3000. Translocation was performed using either a control vector (control condition) or a mouse pLEX-Lcor-HA overexpression construct. The culture medium was changed 24 hours after translocation, and the supernatant was collected at 48 and 72 hours for EV isolation. Cells, fragments, and large vesicles were sequentially centrifuged at 500 g for 10 minutes, 2000 g for 15 minutes, and 10000 g for 30 minutes, followed by ultracentrifugation at 70000 g for 60 minutes in a Beckman Coulter L-90K ultracentrifuge. The EV was first removed by [method]. The resulting EV pellet was resuspended in PBS. The RNA content of the EV was estimated by RNA isolation (Qiagen) and quantification with Nanodrop, and the protein content was measured by bicinchoninate assay. Before the assay, the EV was treated with 10 μg / ml of RNase A (Fisher Scientific, catalog number: 12091021) and 5 μl of [method]. 20 mg / ml Proteinase K (Fisher Scientific, Catalog No.: EO0491) The cells were treated at 37°C for 30 minutes. Cultured 4TO7 cells were treated with 4 ng / μl of EV protein at 37°C for 72 hours. In in vivo metastasis experiments, 50,000 4TO7 cells were injected via TV. BLI imaging indicated metastatic colony formation (2 × 10⁶). 6 If a high photon flux was observed, mice were treated every 3 days by postorbital sinus injection with 10 mg / kg of anti-PD-L1 (atezolizumab, Tecentriq) and 8 μg of control EV or Lcor-HA EV-derived EV protein (in a total volume of 100 μl of PBS). Metastatic lesions were monitored by BLI photon flux, and the lungs were then collected for anti-HA IHC staining and hematoxylin-eosin staining.

[0063] IF, LSFM, and IHC analysis For IF analysis of LCOR-OE and LCOR variants, cells were seeded on coverslips, fixed with methanol at -20°C for 1 hour, and washed with acetone. Samples were blocked with blocking buffer for 30 minutes, incubated with anti-HA at room temperature for 2 hours, washed with PBS, and incubated with the secondary antibody Alexa Fluor 488-anti-rabbit at room temperature for 1 hour. For IF analysis of the LCOR-GFP knock-in reporter system and pan-HLA expression, cells were seeded, fixed with 4% paraformaldehyde (PFA) at room temperature for 15 minutes, washed, and blocked with blocking buffer at room temperature for 1 hour. Cells were incubated with anti-pan-HLA-ABC at room temperature for 2 hours, followed by incubation with Alexa Fluor 647-anti-mouse. Images were acquired using a Nikon Eclipse Ni-E upright fluorescence microscope (Nikon Solutions Corporation), and data were collected using Ni Setup Tool v.1.2.2. Fiji software was used for further analysis.

[0064] For imaging of living organoids transduced with the SORE6+CSC reporter, the inventors used an LS1 live light-sheet microscope system (Viventis). Individual organoids were incubated with anti-pan-HLA-ABC at room temperature for 2 hours, washed 5 times with PBS, and incubated with Alexa Fluor 647-anti-mouse at room temperature for 1 hour. Hoechst33342 (Fisher, catalog number: H3570) was used. 3D rendering of the organoids was performed using FIJI software ClearV I ran it using the olume plugin.

[0065] CD45 + and CD8 +For IHC analysis of T cell infiltration, paraffin-embedded mouse tumor samples were stained with 3,3'-diaminobenzidine (DAB) using Auto Stainer Plus (Dako). Tissue sections (3 μm) were stained with CD8 and CD45 antibodies, and necrotic areas were excluded from quantification. For LCOR evaluation of clinical samples, paraffin-embedded sections (3 μm) from tumor tissue blocks were stained with anti-LCOR at room temperature for 1 hour, followed by ink staining with anti-rabbit Ig-dextran polymer (Flex, Agilent). Incubation was performed. Sections were visualized with DAB and counterstained with hematoxylin. All incubations were performed on the Agilent Link platform. Nuclear immunoassay score ( LCOR expression in samples was evaluated using nIRS. nIRS ranges from 0 to 12 as the product of a positive cell percentage score (0-4) (0: 0%; 1: 1%-30%; 2: 31%-60%; and 3: >60%) and a staining intensity score (0-3) (0: no reaction; 1: weak signal; 2: mild signal; and 3: strong signal). To confirm Lcor-HA delivery to metastatic lesions, lung metastases treated with control or Lcor-HA EV were collected and fixed overnight with 4% PFA. Paraffin-embedded sections (3 μm) from tissue blocks were stained with anti-HA at room temperature for 2 hours. For all IHC analyses performed in this study, slides were visualized and analyzed using either QuPath-0.2.0 (Reference 66) or CellSens software.

[0066] Time-lapse confocal microscopy imaging Stained tumor cells (CellTracker Deep Red; Fisher, catalog number: C34565) are placed on μ-Slide 8-well coverslips (Ibidi, catalog number: C34565). In RPMI medium containing SYTOX green dye (Invitrogen, number: S7020) (G number: 80826), stained CD8 +T cells (CellTracker CM-Dil Dye; Fisher, catalog number: C7000) were co-cultured in a 1:1 ratio for 16 hours. Forty random regions were selected and used in the Zeiss Cell Observer. Analysis was performed using HS (Zeiss) to obtain green, red, deep red, and phase difference channels. Images were acquired every 5 minutes. FIJI software was used to process and visualize the images.

[0067] Virus production and transduction of cell lines HEK293T cells were transfused using lentiviral plasmids along with pocket plasmids (VSVG) and gag-pol plasmids (pCMV-R8.91) according to a standard lentiviral packaging protocol. The cell lines were transduced in 6-well plates using concentrated virus in a medium suitable for each cell line containing 8 μg / ml polyblen, and selected based on corresponding antibiotic resistance.

[0068] ChIP and ChIP-seq library preparation For ChIP-qPCR and sequencing, cells are placed in 150 mm cells. 2 They were grown on plates, fixed, dissolved, and then Bioruptor Pico Tubes (Diagenodes) were used. Used with Sonication Beads (Diagenode), Bioruptor Using a sonicator (Diagenode), I set up seven 30-minute on / 30-minute off cycles to generate ultrasound. Wave-treated. The sample was treated with 5 μg of anti-HA antibody or 5 μg of goat anti-rabbit IgG (R&D, No. The chromatin was incubated overnight at 4°C with rotation, along with either SC-2025. Dynabeads Protein A for Immunoprecipitation (Fisher Scientific) was used for chromatin isolation. Immunoprecipitated chromatin The chromatin was washed, eluted, and purified using the Qiagen DNA Purification Kit (Qiagen). For ChIP-seq, the quality and quantity of chromatin were determined using Agilent's Bioanalyz. I verified this using er. The library was tested on the HiSeq 2500 platform (Illu The sequences were determined using mina (50 base pairs (bp) single-ended). The quality of the fastq files was checked using FastQC software (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ). Reads were aligned with Gencode version 27 (GRCh38 / hg38 assembly) of the Homo sapiens genome (https: / / www.gencodegenes.org / human / release_27.html) using the bowtie2 mapper (reference 69). The quality of the mapped files (BAM format) was checked using QualiMap. Peak 70 in each sample was identified using the MACS2 program (reference 71) (narrow peaks with q<0.1 were selected first). Peak overlap between biological replicates was searched using the Bedtools72 toolset.

[0069] Knock-in fabrication The inventors constructed two different LCOR knock-ins in the endogenous LCOR of human chromosome 10 of MDA-MB-231: GFP-tagged LCOR (LCOR-GFP) and HA(×3)-tagged LCOR (IRES-GFP). The inventors cloned a single guide RNA targeting the 3' end of exon 8 into the pX330-U6 plasmid. Insertion of each guide was confirmed by PCR and Sanger sequencing. To construct donor vectors for LCOR-GFP and LCOR-3×HA-IRES-GFP knock-ins, a 1kb homology arm targeting the cleavage site was amplified and inserted into a homologous recombination repair (HDR) plasmid donor skeleton. Plasmid sequences were confirmed by Sanger sequencing. MDA-MB-231 was simultaneously transfused using lipofectamine 3000 with plasmids HDR-GFP-HDR or HDR-3×HA-IRES-GFP-HDR and pX330-U6-sgRNA in a 10:1 ratio. Cells were collected, and individual cells were transfused with GFP. + We selected the appropriate candidates. Single clones were tested by flow cytometry and Sanger sequencing.

[0070] Molecular cloning and plasmids LCOR overexpression plasmids (control pLEX-MCS and pLEX-HA; pLEX-mouse Lcor and human LCOR-HA, LCOR-LSKAA-HA, and LCOR-ΔHTH-HA) and OVA overexpression plasmid (pLEX-OVA-IRES-mCherry) were provided by Y. Kang's laboratory. pLEX-Lcor-HA was prepared by PCR amplification of complementary DNA of mouse Lcor, addition of HA to the 3' end, and insertion of these together into pLEX-MCS after digestion with SpeI and AgeI (NEB). For gene knockdown assays, shRNAs for targeting the mouse gene Lcor (number: TRCN0000085107) and the human gene LCOR (numbers: TRCN0000016306 and TRCN0000436034) were prepared using Sigma-Aldrich or The plasmids used were purchased. Other plasmids used included the SORE6 series 31, which contains minCMV-GFP, minCMV-RFP, SORE6-minCMV-GFP, and SORE6-minCMV-RFP, as well as the pQCXIN / ZsGreen proteasome activity reporter 46. The ISRE reporter was constructed by introducing the ISRE sequence (interferon-stimulated response element (ISRE): 6 tandem repeats of 5'-CAGTTTCACTTTCCC-3') before the CMV minimal promoter (mCMVp)-RFP after removing the binding site of the SORE6-minCMV-RFP construct using BstZ17I-HF and ClaI(NEB) restriction enzymes. Mutant versions of the ISRE reporter were also designed by mutating two thymines in two highly conserved thymine triplets to guanine. Sequence insertions were evaluated by Sanger sequencing.

[0071] Flow cytometry analysis and cell sorting Flow cytometry analysis data was collected using either a Fortessa Flow Cytometer (BD) or LSRII Flow Cytometry (BD) with FACSDiva software v.9.0, and FlowJo software v. Analysis was performed according to 10.8.1 (FlowJo). Cells were filtered through a 70 μm filter, counted, diluted in PBS + 10% FBS buffer, and then in cell lines or organoids, were stained with anti-SIINFEKL (OVA), anti-mouse or human PD-L1, anti-TAP1 intracellular staining, proteasome activity using the pQCXIN reporter system 46, pan-HLA-ABC, anti-mouse or human β2M, pan-H2-Kd / Dd, CSC SORE6 reporter 31, and ISRE reporter; and OT-1 CD8 +For T cells, a standard staining protocol using anti-CD69 was followed. For the analysis of tumor-infiltrating lymphocytes, the tumor was isolated as described above. The cells were filtered through a 70 μm filter, counted, diluted in PBS + 10% FBS buffer, and then stained using anti-CD45, anti-CD3, anti-CD8, anti-CD4, anti-CD11c, anti-CD11b, and anti-F4 / 80. For cell cycle analysis, cells were fixed in ice-cold methanol at 4°C for 2 hours, stained with propidium iodide buffer, and DNA content was analyzed by flow cytometry.

[0072] Using either a FacsAria (BD) or Influx (BD) cell sorter, CD24 lo / CD44 hi CSC populations were isolated from PDO and then incubated with anti-CD24 and anti-CD44. Cell sorting from cell lines involved washing cells with PBS, trypsinizing, counting, and diluting. AT3 and Py8119 cells, which stably express OVA and are low and high immunogenic cells, were incubated with anti-OVA. - / low and OVA high The population was gated and isolated. For validation of the SORE6 system in our mouse model, Py8119 and AT3 cells were converted to GFP. + and GFP - Cells were isolated. For CSC isolation from MDA-MB-231, cells were stained with anti-CD44 and anti-CD104, and CD44 hi / CD104 hi Enriched CSC population and CD44 hi / CD104 lo We selected a non-enriched CSC population and used the Aldefluor Kit (Stemcell technologies). ) using ALDH - ALDH + Compared to ALDH, gated using DEAB control according to the manufacturer's instructions for use, + The group was divided. HMLE cells were marked with CD44. hi / CD24lo Enriched CSC population and CD44 lo / CD24 hi A selection was made from 43 non-enriched CSC populations. In CSC isolation using cell lines 4TO7 and AT3, CD24 hi / CD29 hi CSC population and CD24 lo / CD29 lo Non-CSC population, and CD44 hi / CD24 lo CSC population and CD44 lo / CD24 hi Non-CSC populations were isolated. Cells were incubated with anti-CD29, anti-CD24, and anti-CD44.

[0073] RT-qPCR analysis Total mRNA was purified using the RNeasy Mini Kit (Qiagen) and reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies). RT-qPCR was performed using the Light Using tCycler 480 SYBR Green I Master (Merck) The process was executed. Data was collected using QuantStudio 12K Flex software.

[0074] RNA-seq analysis RNA was isolated from tumors using the Qiagen RNA extraction kit. Poly-A sequencing was chosen for library preparation, and samples were sequenced at the Centre of Genomic Regulation (CRG) using an Illumina Hi-Seq 2500 platform at a setting of 1 × 50 bp. Raw data (fastq files) were quality-checked using FastQC software (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ). Ribosomal RNA was estimated from the raw data using riboPicker74. Raw reads were aligned with the Mus musculus genome (Gencode release M24 of the GRMm38 / mm10 assembly: https: / / www.gencodegenes.org / mouse / release_M24.html) using STAR Mapper 75. Raw read counts for each gene and sample. Gene counts were obtained using STAR (quantMode TranscriptomeSAM GeneCounts option). Differential expression between experimental groups was evaluated using the R / Bioconductor DESeq2 package v.1.30.1 (Wald test + false positive rate (FDR) correction). Genes with a total raw count of less than 1 across all samples were excluded. A gene was considered differentially expressed if the fold change of the absolute value log2 was greater than 1 and the adjusted P-value was less than 0.05. Using these parameters, the inventors selected the top 300 genes that were upregulated in IRT cells compared to control cells to generate ICB resistance signatures.

[0075] Clinical Dataset Analysis Clinical evaluation of LCOR expression was performed using RNA-seq transcriptome data from the TONIC trial (number: EGAS0001003535) 40 (this trial included samples from 53 patients with metastatic TNBC who had received prior induction therapy, with 9 responders (1 complete response and 8 partial responses) and 44 non-responses (1 stable disease and 43 disease progression)); and the I-SPY2 trial (number: GSE173839) 52 patients with stage I-III prior treatment. The analysis was performed using RNA-seq transcriptome data from TNBC samples (treated with durvalumab and olaparib in combination) (9 response cases and 12 non-response cases) and RNA-seq transcriptome data (number: phs001919) 76 from 30 metastatic melanoma biopsies treated with either pembrolizumab or nivolumab (including 13 patients with disease progression, 3 with stable disease, 10 with partial response, and 2 with complete response). The METABRIC dataset was obtained from the open-access site cBioportal (http: / / www.cbioportal.org / index.do), and mRNA expression data from 186 TNBC patients were analyzed. METABRIC data from the TNBC and TONIC trials were also used in GSEA and GSVA.

[0076] GSEA, GO, and Chea Transcriptome and ChIP-seq data generated in this study were submitted and analyzed for GO and ChEA. In the GO analysis, genes that were upregulated and downregulated in IRT cells, as well as promoter / enhancer regions as determined by LCOR ChIP-seq, were investigated for enriched pathways using BioCarta signatures. For ChEA77, the upregulated genes in IRT cells were investigated for enriched transcription factors.

[0077] We analyzed signature correlations in stratified datasets using GSEA, performed 1000 random substitutions of labeled phenotypes, and obtained P-values, q-values, and normalized enrichment scores (NES). Genes were ranked using the formula: r(g)=sign(FCg)(1-Pg), where r(g) is the enrichment score for each gene, FCg represents the median multiplier change (FC) between the two conditions, and Pg represents the p-value of the Wilcoxon rank-sum test 40. The following datasets were used: METABRIC (samples were stratified as higher or lower than LCOR expression levels based on median expression after normalization was confirmed); and TONIC dataset 40 (stratified by response and non-response cases). IRT transcriptome data were also compared with IRT control cells. The dataset was investigated using the following different signatures: a publicly available embryonic stem cell-like signature (BENPORATH_ES_1) containing 379 genes; and LIU mammary ER containing 164 UP genes satisfying more than 2FC. - CSC Signature (CD24 from HCC1954, MC1, SUM149, and SUM159 cells) - / CD44 + ER isolated by - APM Human Kyoto Encyclopedia of Genes and Genomes (KEGG:M16004) signature containing 34;88 APM-related genes (generated by median expression of mammary CSCs); GO:0048002 mouse APM signature containing 76 APM-related genes; the inventors ICB resistance signatures of 300 generated UP genes (greater than 1FC and adjusted P value > 0.05); and previously described immune resistance signatures in melanoma patients 53.

[0078] GSVA Using the GSVA package, we established enrichment scores representing signature enrichment across 78 patients. GSVA was applied using the following datasets: IRT transcriptome RNA-seq data, and METABRIC TNBC clinical data, and 39 ER samples from CCLE. - Transcriptome data of breast cancer cell lines. The dataset was investigated using the following gene signatures: BENPORATH_ES_1 (Reference 29); NOS (NANOG, OCT4, SOX2) targets 29; LIU breast ER - CSC34; APM signature (KEGG:M16004); our IRS; and immunotherapy resistance signature for melanoma (Jerby-Arnon)53.

[0079] Deconvolution and immunophenotypic analysis For the estimation of infiltrating immune cells, the xCell algorithm (https: / / xcell.ucsf.edu / )50 was used for METABRIC TNBC data. Non-hierarchical k-means cluster analysis of four clusters based on Euclidean distance was applied to evaluate LCOR expression in various clusters or immunophenotypes.

[0080] Statistical analysis and reproducibility Statistical methods were not used to pre-determine sample size in animal studies; sample size was derived from pilot studies and previous research. Prior to infusion time, randomization was performed among littermates using female animals of similar age. Researchers were not blinded to their assignments during the experiment and outcome evaluation, as they needed to know which group was being treated. In vivo tumorigenesis assays were monitored blinded by trained technicians. In the in vitro experiments, randomization was unnecessary as statistical methods were not used to pre-determine sample size and all samples were analyzed similarly. Tissue staining scores were determined by three independent researchers blinded to sample information. Independent biological replicates are shown in the figure captions for all in vivo and in vitro experiments. Each result is expressed as mean ± standard error. For all experiments, normality and homogeneity of variance were confirmed by the Kolmogorov-Smirnov test and the Bartlett test, respectively. Statistical significance was determined by either a one-sided or two-sided independent t-test or a paired Wilcoxon signed-rank test for parametric results, and by Welch's t-test for nonparametric results. For multiple independent groups, either a two-way / one-way analysis of variance (ANOVA) or the Mann-Whitney test was applied. The tests used to confirm statistical differences are indicated in the figure captions. For TIC, ELDA software was used in conjunction with Pearson's χ² test. The significance of correlations was evaluated using p-values ​​and the ρ index. For survival analysis, the P-log rank test was applied using GraphPad Prism. All other statistics were calculated using the R and R-studio interfaces (https: / / www.r-project.org / ). Unless otherwise specified, all experiments were replicated at least three times in independent biological experiments.

[0081] result ICB resistance arises from a MaSC-like state. To understand breast cancer ICB resistance, the inventors created a preclinical immunoassay syngeneic model of anti-PD-L1 ICB resistance in vivo. Mouse breast cancer 4TO7 cells were orthotopically transplanted into breast fat pads (MFPs). The tumor size was 0.5 × 0.5 cm. 2 Once the tumors reached a certain size, the mice were treated with anti-PD-L1. After an initial transient response, all tumors developed resistance to anti-PD-L1 treatment (Figure 1a). Anti-PD-L1 immunotherapy-resistant tumors (IRTs) were collected, cultured, and their resistance was reconfirmed in vivo.

[0082] Transcriptome analysis of IRT cells revealed the loss of APM, one of the most downregulated gene ontology (GO) pathways, followed by the expected loss of IFN signaling (Figures 1b and 1c). Interestingly, we discovered enrichment of stem cell signatures within the upregulated pathways (Figure 1b), and using gene set variation analysis (GSVA), we confirmed significant enrichment of CSC-like signatures associated with breast cancer malignancy, e.g., ES_1, NOS_Targets29, and Breast_CSCs (Figure 1d). Furthermore, chromatin immunoprecipitation (ChIP)-enrichment analysis (CHEA) assessed that stem cell transcription factors (SC-TFs) were the most enriched in IRT. The inventors confirmed these transcriptome findings by quantitative PCR using reverse transcription (RT-qPCR), which showed that in IRT cells, the SC-TF genes Pou5f1(Oct4), Sox2, Sox9, and Nanog were enriched, and mammary gland differentiation factors, particularly Lcor, were depleted (Figure 1e). Importantly, IRT cells were found to be rich in CD24, a MaSC marker. + / CD29 hi An increase in this population was also observed, and this population has been reported in 4TO7 cells, etc. (Figure 1f). The selection of these cells by ICB treatment was not observed when we inhibited antigen presentation using β2M knockout (KO) cells, which is consistent with the lack of selective immune-mediated killing. Notably, IRT cells maintained high PD-L1 expression, suggesting resistance to anti-PD-L1 despite expressing the target. We propose that this resistance arose from the downregulation of APM in CSCs, as suggested by the lack of enrichment of CSC-like cells in β2M KO cells after treatment. Further characterization is that IRT cells showed no differences in the cell cycle and had similar proliferation rates to control conditions, while IRT and CD24 hi / CD29 hiBoth CSC systems demonstrated enhanced tumor cell formation ability in the globular generation in vitro. To evaluate the characteristic TIC frequency of CSCs in vivo, the inventors performed orthotopic MFP injection using the limiting dilution method (LDA) into NOD-SCIDγ (NSG) immunodeficient mice. IRT cells were enriched more than 10-fold in TIC frequency (Figure 1g), confirming that CSC characteristics are selected by ICB therapy.

[0083] Next, the inventors performed an in vitro cytotoxic T lymphocyte (CTL) assay by co-culturing AT3-OVA cells and OT-I CD8 T cells that specifically recognize the OVA peptide (SIINFEKL). After 3 days of co-culturing, surviving tumor cells that evaded immunokilling showed CD24, particularly after anti-PD-L1 treatment. lo / CD44 hi AT3 CSCs were enriched (Figure 1h). Therefore, the selected AT3 and 4TO7 CSCs showed resistance to immune-mediated cytotoxicity compared to non-CSCs when co-cultured with OT-I CD8 and JEDI CD8 T cells, respectively (Figure 1i). AT3 CSCs induced weaker antitumor T cell activity compared to non-CSCs. The inventors applied the SORE6 CSC reporter system, which reports SOX2 and OCT4 activity, to AT3-OVA and Py8119-OVA cells. The inventors first verified the fidelity of SORE6 in their model, and then SORE6 + The study showed that when SC-TF was expressed and injected into MFP with LDA, it had a more than 6-fold higher TIC frequency in vivo. Similar to previous results, the surviving cell population in the CTL assay was SORE6 - Selective removal of cells leads to SORE6 + CSCs were highly concentrated, and this effect was further enhanced by anti-PD-L1 treatment. Overall, these results demonstrate the ability of the CSC population to evade anti-PD-L1 ICBs.

[0084] Although there are limitations to studying immune-specific interactions in human models, CD104 isolated from MDA-MB-231 cellshi / CD44 hi CSCs showed stronger resistance to anti-PD-L1 when co-cultured with human peripheral blood mononuclear cells (PBMCs) in an allogeneic in vitro environment. Furthermore, MDA-MB was introduced into immunohumanized mice using PBMCs. When -231 cells were injected orthotopically, after 5 weeks, tumors treated with anti-PD-L1 showed CD104 hi / CD44 hi The CSC population was enriched.

[0085] LCOR low Breast CSCs have reduced antigen-presenting ability. Based on the finding that ICB-resistant cells exhibit a stem cell-like phenotype and low APM expression (Figure 1b, Figure 1c), we analyzed the available MaSC transcriptome profiles. As expected, fetal mammary stem cells (fMaSCs) showed simultaneous downregulation of APM pathway genes, including immunoproteasome factors (PSMB8 and PSMB9), transporters (TAP1 and TAP2), β2M, and MHC-I genes.

[0086] To study the APM pathway in human CSCs, the inventors prepared patient-derived organoids (PDOs) from clinical samples and extracted CD24 from four different TNBC patients. lo / CD44 hi CSC37 was isolated (Figure 2a). Our analysis, including RT-qPCR analysis (Figure 2a) and flow cytometry of pan-HLA-ABC and β2M (Figure 2b), revealed that CD24 lo / CD44 hi Lcor low Downregulation of the APM gene was confirmed in CSC clinical samples. Furthermore, three-dimensional (3D) PDO imaging using advanced light-sheet fluorescence microscopy (LSFM) revealed SORE6 + The absence of pan-HLA staining in CSCs was also demonstrated (Figure 2c). HMLE was also observed in human mammary gland cell lines. CD24 lo / CD44 hi Stem cells and MDA-MB-231 CD104 hi / CD44 hi The CSC population, non-CSC and ALDH + Compared to the population, the APM gene and Lcor were downregulated. Accordingly, mouse 4TO7 CSCs and IRT cells also showed low Lcor and low APM expression. Next, MDA-MB-231 cells were generated with knock-in of fused LCOR-green fluorescent protein (GFP) to report the endogenous protein levels and localization of LCOR. As expected, LCOR low MDA-MB-231 cells are OCT4 + / SOX2 + CSC(SORE6 + ) was concentrated. Next, the inventors isolated the cells based on GFP (LCOR-GFP) levels using fluorescence-activated cell sorting (FACS) and performed RT-qPCR on LCOR - / low Cells and LCOR high The expression of the APM gene in cells was measured. APM gene expression gradually increased with increasing LCOR levels (Figure 2e). This was further confirmed by immunofluorescence (IF) of pan-HLA-ABC and LCOR-GFP (Figure 2f). These results are based on human LCOR low This shows reduced APM activity in CSCs.

[0087] To demonstrate APM impairment in CSCs, breast cancer cells were isolated based on APM activity using OVA antigen peptide presentation as a readout. The inventors ectopically expressed native full-length chicken egg ovalbumin (OVA) in AT3 and Py8119 cells, which are H2-K1b haplotype cells established from mouse breast cancer C57BL / 6J PyMT tumor 44. Ectopic OVA was processed by the immunoproteasome to produce the OVA257-264 (SIINFEKL) peptide, which is transported and presented in the context of MHC-I H2-K1b. As hypothesized, the isolated OVA... - / low Cells are measured by RT-qPCR, OVA highexpressed higher levels of CSC genes (Oct4, Sox2, Sox9, and Nanog) compared to cells and significantly lower levels of Lcor (Figure 2h). Thus, by flow cytometry, CD24 lo / CD44 hi AT3 CSC population was mostly separated into OVA - / low AT3 cells (Figure 2i), while CSCs were only partially separated by Pd-L1.

[0088] Functional assays in both cell lines demonstrated enhanced tumorsphere formation ability and enhanced TIC ability of OVA - / low cells in vivo in orthotopic MFP LDA in NSG immunodeficient mice (Figure 2j). This reflects the inherent tumor-initiating stem cell ability of OVA - / low cells.

[0089] Therefore, in tumor cell heterogeneity, LCOR low CSCs have a defective APM system as an important immune evasion property for ICB avoidance. LCOR regulates APM independently of interferon signaling. LCOR is one of the most downregulated mammary differentiation factors in our IRT model, and LCOR low CSCs are associated with attenuation of APM. Here, we show the expression correlation of LCOR and APM components in estrogen receptor (ER)-negative breast cancer cell lines of the Cancer Cell Line Encyclopedia (CCLE) (Figure 3a). Furthermore, patient stratification of the TNBC METABRIC dataset based on LCOR levels demonstrates a strong correlation between LCOR expression and both the APM pathway (Kyoto Encyclopedia of Genes and Genomes (KEGG): M16004) (Figure 3b) and IRS (Figure 3c). These results suggest that LCOR may play an important role in APM regulation.

[0090] To investigate the mechanistic relationship between LCOR and the APM pathway, the inventors used gain-of-function and loss-of-function modifications of LCOR in MDA-MB-231 and HMLE cells. Notably, ectopic LCOR expression induced APM pathway genes in both cell types, while LCOR-KD reduced their expression (Figure 3d). Furthermore, the inventors measured the expression of different APM components by flow cytometry in these models. LCOR-KD cells mimicked the phenotype of CSCs and showed attenuation of APM, as determined by attenuation of the proteasome activity reporter pQCXIN / ZsGreen, and decreased expression of the transporter (TAP1) and presentation molecules (β2M and HLA-ABC) (Figures 3e-3g). The opposite result was obtained in LCOR-OE, which enhances antigen processing and presentation (Figures 3d-3g).

[0091] Surprisingly, these effects were independent of IFN stimulation. Although IFNγ treatment enhanced the effect of LCOR on APM components, ruxolitinib, an inhibitor of JAK1 / JAK2-mediated STAT / IRF activation, did not affect LCOR's ability to induce different APM components in vitro (Figures 3e-3g). Importantly, the effect of IFNγ treatment was abolished in LCOR-KD cells. This suggests that LCOR levels modulate sensitivity to IFN and the effect of IFN on the APM pathway (Figures 3e-3g). To examine a wide range of APM activity, mouse AT3-OVA Lcor-KD cells and Py8119-OVA Lcor-KD cells showed attenuated OVA presentation, while Lcor-OE cells showed high presentation under all three conditions, thus demonstrating the essential role of LCOR in the regulation and initiation of APM activity (Figure 3h). Similarly, IFNγ treatment failed to enhance OVA presentation in Lcor-KD cells. Overall, our data demonstrate that LCOR levels determine APM activity in tumor cells, with or without IFN signaling, highlighting the dominant role of LCOR in antigen presentation.

[0092] LCOR directly modulates APM elements via ISRE binding. To understand how LCOR modulates the APM pathway, we transduced ectopic expression of LCOR, as well as mutant LCORs containing a double-point mutation in the nuclear receptor (NR) binding domain that prevents binding to NR (LSKLL → LSKAA) and a deletion in the HTH DNA binding domain that blocks putative binding to DNA (ΔHTH), into MDA-MB-231 cells. We confirmed appropriate overexpression and nuclear translocation of each variant. Next, we performed chromatin immunoprecipitation sequencing (ChIP-seq) analysis. In wild-type and LSKAA-type LCOR, the highest peak cluster across the entire genome was found on the short arm of chromosome 6 (chr6), but not in the ΔHTH mutant (Figure 4a). Importantly, this approximately 8 Mb genomic region (chr6:27810120~35980577) The LCOR cluster contains all APM genes except β2M, which is located on the MHC-I cluster and chr15 (Figures 4a and 4b). Consistent with this, gene expression analysis of MDA-MB-231 cells showed that wild-type and LSKAA-type LCOR induce APM genes, but ΔHTH mutants do not (Figures 4a and 4b). Notably, the LCOR peaks were located at the gene regulatory elements (GREs) of these genes, including β2M (Figure 4c). This suggests direct master control of the APM cluster, as also confirmed by ChIP-qPCR of endogenous LCOR using the LCOR-HA knock-in system. Accordingly, enrichment analysis of the ChIP-seq peaks ranked the APM pathway as the most highly enriched pathway among BioCarta's biological processes (Figure 4d). Interestingly, analysis of available transposase-accessible chromatin sequencing assay (ATAC-seq) data revealed that this region is shut down in fMaSCs. This reflects a conserved gene regulatory mechanism of mammary gland cell immunogenicity. Therefore, we investigated the evolutionary conservation of LCOR in other species and found that LCOR, particularly the NR domain and HTH domain, is highly conserved in all vertebrates, with the latter being the only domain conserved beyond vertebrates. Thus, it is an ancient DNA-binding domain originating in prokaryotes as a transcription factor domain. These findings support the conserved role of LCOR through its HTH domain in regulating APM transcription.

[0093] Our transcriptome and conservation analyses suggest that LCOR may act as a transcriptional activator. However, LCOR has previously been described as a transcriptional corepressor, and its function as a candidate transcriptional activator had not yet been demonstrated. By performing motif discovery analysis of LCOR ChIP-seq peaks using HOMER software, we identified ISRE, particularly the interferon regulator 1 (IRF1) binding site typical of interferon-inducible genes and APM genes in antiviral responses, among the top predicted motifs (Figure 4e). To demonstrate that LCOR can activate APM genes, we created a promoter-reporter with six tandem repeats of the ISRE motif (6×CAGTTTCACTTTCCC) upstream of the minimal CMV promoter, and a similar mutant version (6×CAGTGGCACGGTCCC), to identify specific LCOR binding (Figure 4f). As quantified by red fluorescent protein (RFP) flow cytometry in MDA-MB-231 cells, only LCOR and LSKAA increased reporter activity, while ΔHTH did not (Figure 4g). Notably, no induction was detected when the ISRE sequence was mutant (Figure 4g). This indicates that LCOR binds to ISRE and activates transcription. Next, the inventors combined the ISRE reporter with LCOR-GFP knock-in and created a unique flow cytometry system to study ISRE activity dependent on endogenous LCOR regulation. The inventors investigated the effects of different conditions, including IFNγ treatment and different IFN inhibitors (ruxolitinib (JAK1 / JAK2 inhibitor) and BX-795 (TBK1 inhibitor that inhibits STING signaling)), to further elucidate the interference of IRF and LCOR on reporter activity. The inventors found that IFNγ treatment affects LCOR + In cells (moderate and high), ISRE activity was increased, but LCOR - We observed that it did not increase in cells. Furthermore, ruxolitinib was found to be effective in LCOR - It inhibits ISRE activity only in cells, and LCOR +No inhibition occurred in the cells. This indicates that LCOR-mediated ISRE induction is independent of IFN and essential for the transcriptional activation of ISRE-regulated genes (Figure 4h). Therefore, these treatments did not suppress ISRE induction in LCOR-OE cells, and IFNγ failed to induce ISRE in LCOR-KD cells. This is consistent with the independent induction of APM activity (Figures 3e-3h).

[0094] The inventors further confirmed the transcriptional activity of LCOR by performing RNA polymerase II subunit B (POLR2B) ChIP. POLR2B bound to the MHC-I gene only in the presence of LCOR and not in LCOR-KD cells. Furthermore, POLR2B was enriched in LCOR-OE or LSKKA-OE cells but not in ΔHTH mutants, suggesting the necessity of LCOR binding to the MHC-I gene promoter. Overall, these results reveal that LCOR is a transcriptional activator of the APM pathway.

[0095] LCOR promotes immune infiltration and killing of tumors. Next, we investigated the effects of LCOR-mediated APM induction on tumor immunity. AT3-OVA and Py8119-OVA cells were CD8 + OT-I T cells co-cultured in In vitro CTL assay, and 4TO7-EGFP cells are CD8 +The in vitro CTL assay co-cultured with JEDI T cells showed enhanced immune killing of Lcor-OE cells in both systems (Figure 5a, Figure 5b). Knockdown and knockout of β2m in Lcor-OE cells rescued survival rate and avoidance of T cell killing, demonstrating that this effect depends on antigen presentation ability (Figure 5c). In addition, Lcor-KD cells avoided immune-mediated cell killing, consistent with the decreased OVA presentation ability (Figure 3h) (Figure 5d). Lcor-OE cells enhanced T cell activation as measured by CD69, while Lcor-KD cells reduced it (Figure 5e). In the tumor growth experiment, 4TO7 Lcor-OE cells showed stronger Lcor-mediated reduction in immunocompetent mice (Balb / c) than in NSG immunodeficient mice. This suggests the induction of an immune response due to immunogenicity mediated by LCOR. The opposite effect was true for Lcor-KD tumors. 4TO7 Lcor-OE tumors showed a significant increase in infiltrating CD4 and CD8 lymphocytes compared to control tumors as measured by immunohistochemistry (IHC) and flow cytometry (Figure 5f, Figure 5g), + and also showed a non-significant increase in white blood cells (Figure 5f). + / CD3 - The compartment showed no significant changes in dendritic cells (CD45 + / CD3 - / CD11c + / F4 / 80 - ) and macrophages (CD45 + / CD3 - / CD11b + / F4 / 80 + ). These results support a lymphocytic immune response against them due to the high immunogenicity of Lcor-OE tumors.

[0096] The inventors performed deconvolution analysis of the METABRIC dataset using xCell50 to estimate the immunocomposition of 186 samples from 51 TNBC patients. The inventors generated the following immunophenotypic clusters depicting the immune landscape: Cluster 1: Low-invasion immunocellular tumors; Cluster 2: Immunosuppressive population; Cluster 3: Cytotoxic population; and Cluster 4: Highly cytotoxic population. Next, the inventors analyzed LCOR levels in these different clusters and found higher expression in the cytotoxic cluster (Figure 5h, Figure 5i). LCOR high The tumors were also more enriched with CD4, CD8, and γδT lymphocyte gene signatures. Overall, these results support the premise that LCOR promotes immunogenic and adaptive immune infiltration in TNBCs and mediates antitumor immunity.

[0097] LCOR levels and ICB response in TNBC patients The inventors performed LCOR IHC analysis on a small set of matched TNBC clinical samples before and after ICB treatment in a neoadjuvant setting. Two patients (P1 and P2) were treated with anti-PD-L1 atezolizumab plus multi-agent chemotherapy (Abraxane and carboplatin), while the other two (P3 and P4) were treated with anti-PD1 nivolumab plus a SYK / FLT3 inhibitor. In all cases, LCOR expression in residual lesions was lower (Figure 6a). These data support the hypothesis that LCOR cells are eliminated by neoadjuvant ICB combination therapy.

[0098] The inventors also investigated the importance of LCOR in response to ICB in larger clinical trials. An analysis of the TONIC trial, which included 40 pre-treatment metastatic TNBC patient samples from 53 patients, showed higher levels of LCOR in response patients (Figure 6b). In addition, data from the Phase II I-SPY2 trial of 52 TNBC patients using durvalumab, olaparib, and neoadjuvant paclitaxel also showed higher levels of LCOR in response patients (Figure 6b). These findings demonstrate that LCOR levels are associated with the response to combination therapy including ICB in TNBC. To evaluate whether these findings apply to other cancer types, the inventors investigated a melanoma cohort treated with anti-PD-1 monotherapy, which showed higher levels of LCOR in response patients. Furthermore, the inventors' breast cancer IRS overlapped with the ICB resistance signature 53 of melanoma, and LCOR high TNBC patients showed an inverse correlation with the latter signature. Overall, these results support the association between LCOR and ICB clinical efficacy.

[0099] LCOR overcomes resistance to ICB and leads to tumor eradication. Antigen presentation and PD-L1 are characteristic features of the response to anti-PD-1 / PD-L1 therapy. LCOR slightly increased PD-L1 expression in 4TO7 and AT3 cells and showed a positive correlation in TNBC patients. This can be explained by the fact that LCOR may stimulate IFN sensitivity and induce PD-L1 (References 3, 5). Notably, the combination of positive levels of PD-L1 and potent APM induction by LCOR creates an ideal tumor environment for anti-PD-1 / PD-L1 therapy. Therefore, we performed orthotopic MFP transplantation of syngeneic 4TO7 cells into immune-responsive mice, and the tumor was reduced to 0.5 × 0.5 cm². 2The tumors were allowed to reach a certain size, and then weekly anti-PD-L1 ICB therapy was initiated. While untreated control tumors and control-treated tumors continued to grow, Lcor-OE tumors treated with anti-PD-L1 ICB completely regressed, and complete response (CR) was observed in all mice by day 20 (Figures 6d, 6e). Importantly, CD4 / CD8 compartment depletion did not result in a response to ICB, confirming that the CR observed with anti-PD-L1 in Lcor-OE tumors was mediated by the adaptive immune system. We performed up to five independent experiments and observed CR in 49 out of 50 Lcor-OE tumors (Figure 6f). Notably, the only Lcor-OE tumor that did not respond had lost ectopic expression of Lcor. All 49 mammary glands that showed CR were tumor-free two months after discontinuation of anti-PD-L1 administration. Fifteen of these mice were followed for one year, and none experienced tumor recurrence; all mice had tumor-free mammary glands. This suggests that the inventors irreversibly eradicated the tumors and cured these mice (Figure 6f, Figure 6g). Accordingly, 4TO7 Lcor-KD tumors showed stronger resistance to anti-PD-L1 compared to control 4TO7 cells (Figure 6h). In the AT3 syngeneic model in C57BL / 6J mice, complete response (CR) of Lcor-OE tumors with ICB therapy was confirmed both in vivo and in vitro.

[0100] To study the dominant role of LCOR beyond IFN signaling in vivo, the inventors conducted MFP experiments comparing the effects of Lcor-OE and the type I IFN inducer poly(I:C)54. The combination of poly(I:C) treatment and anti-PD-L1 did not achieve the same efficacy as the Lcor + anti-PD-L1 condition, further demonstrating the dominant role of LCOR in ICB therapy beyond IFN-mediated effects.

[0101] Next, the inventors performed a preclinical lung metastasis assay using 4TO7 tail vein (TV) administration to enable the establishment and growth of lung metastases before initiating anti-PD-L1 therapy (Figure 6i). To achieve synchronized metastasis between different conditions, the inventors injected them three times more often due to the reduced tumorigenic potential inherent in Lcor-OE cells. After 4 weeks of anti-PD-L1 treatment, 4TO7 Lcor-OE metastases were cured in 5 out of 6 mice, while against All of Iruru's metastatic tumors progressed despite anti-PD-L1 exposure (Figure 6i). Overall, these preclinical assays demonstrate a definitive curative effect of anti-PD-L1 treatment, mediated by the effect of LCOR on the immunogenicity of tumor cells, making it a promising therapeutic target for early and advanced TNBC.

[0102] In vivo mRNA-based LCOR therapy combined with ICB Therapeutic mRNA delivery using nanoparticles has enabled the rapid development of highly effective vaccines against COVID-19 and has the potential to revolutionize cancer treatment. Based on current knowledge of extracellular vesicles (EVs) and mRNA delivery, we designed a proof-of-concept approach to restore Lcor expression by introducing Lcor mRNA into tumor cells and combining this treatment with ICB. We ectopically expressed HA-tagged Lcor in HEK293T cells. These cells produce a large number of EVs containing ectopic Lcor-HA mRNA transcripts (Figure 7a). 4TO7 cells treated in vitro with Lcor-HA mRNA EVs showed uptake and translation of the Lcor protein, as detected by immunoblotting with anti-HA (Figure 7b), and exhibited modified nuclear localization (Figure 7c). Lcor EVs reduced the CSC population, upregulated the APM gene, and CD8 in 4TO7 cells. +T cell-mediated killing was enhanced. Next, the inventors designed a preclinical lung metastasis assay to test Lcor mRNA therapy. As proof in vivo, 5 days after EV administration, lung metastases already showed uptake by most tumor cells and translation of Lcor-HA protein (Figure 7d). Next, mice sequentially treated with a combination of EV-based Lcor mRNA therapy and anti-PD-L1 showed significantly longer survival and complete clearance of lung metastases compared to EV controls and anti-PD-L1 therapy (Figures 7e-7g). These results suggest that LCOR mRNA therapy could be a potential therapeutic partner to ICB therapy.

[0103] Function of LCOR in ER+BC Estrogen receptor-positive breast cancer cells (ER) that ectopically express LCOR + Co-culture assays of MCF7 cells (BC) do not show a highly efficient killing effect mediated by immune cells. In this example, allogeneic systems using human peripheral blood mononuclear cells (PBMCs) were used for 5 days with and without anti-PD-L1 (atezolizumab) (Figure 8). Conversely, ectopic overexpression of LSKAA, which lacks the ability to interact with ER (and other NRs), was more efficient, and the inventors observed potent killing of MCF7-LSKAA tumor cells mediated by the immune system. This result mimics LCOR in TNBC tumors where ER is absent and therefore LCOR's mediation of APM gene expression is not hindered by interaction with ER. These results provide functional evidence that LCOR fragments that cannot interact with NRs have far superior efficiency in inducing immunogenicity in tumor cells.

[0104] LCOR and LCOR variants / fragments that induce AMP in different breast cancer subtypes MCF7(ER) transduced with LCOR and the mutant LSKAA +Using ), the inventors measured APM gene expression by qPCR. Induction of APM gene expression was observed only when LSKAA was used (Figure 9). Furthermore, LSKAA was found in MCF7, MDA-MB-231 cells (ER - APM can be induced to a level comparable to that of LCOR in ). These results clearly demonstrate how much more efficiently LSKAA increases APM gene expression by avoiding interaction with NR.

[0105] Interestingly, ChIP assays performed to investigate specific LCOR binding to APM gene regulatory elements (promoter, promoter, etc.), as shown in MDA-MB-231 (Figure 4), revealed how MCF7(ER) + This clearly shows that direct binding of LCOR and regulation of AMP do not occur in ) cells. However, LSKAA directly binds to the APM gene by disrupting its interaction with NR, and regulates... This can be explained (Figure 10). These results further support the specificity of this mechanism and the high efficiency with which LSKAA induces APM gene expression.

[0106] Flow cytometry analysis shows how LCOR can consistently induce HLA genes in ER-negative BCs such as MDA-MB-231 (Figure 3), but LCOR is ER + BC cells do not allow for very potent induction. Instead, LSKAA can potently increase MHC-I molecules (HLA and β2M) as measured by flow cytometry (Figure 11). Importantly, LSKAA significantly increases the abundance of HLA molecules in MDA-MB-231 cells compared to LCOR wild-type cells (Figure 11). This suggests that LCOR is ER - Although a strong APM response can be induced in BC, we demonstrate that LSKAA is even more efficient in doing so.

[0107] Therefore, additional shorter versions of LCOR (referred to herein as variants or fragments) were constructed and functionally tested (Figure 12). The results clearly demonstrate how much shorter versions of this protein still possess the activity to induce APM genes, as measured by flow cytometry such as HLA-ABC, and that the smallest functional element is the HTH domain. The delta-HTH (ΔHTH) fragment does not have the ability to induce HLA, while the remaining fragments induce HLA in MDA-MB-231 cells (Figure 13). Importantly, the most efficient fragments are the longest fragments without a complete NR, namely LSKAA and the delta-NR fragment (Figures 12 and 13).

[0108] Item(CLAUSES) 1. A variant or fragment of a ligand-dependent corepressor (LCOR) that is isolated or synthesized, or a polynucleotide sequence encoding such a variant or fragment of a ligand-dependent corepressor (LCOR), characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) includes the amino acid sequence SEQ ID NO: 3, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO: 3, and at least one NLS motif (nuclear localization signal) of the amino acid sequence SEQ ID NO: 4, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO: 4, and further characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) is lacking or disrupted / inactivated a nuclear receptor-binding domain.

[0109] 2. A variant or fragment of a ligand-dependent corepressor (LCOR) as described in item 1, characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) consists only of a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 5, or substantially identical thereto.

[0110] 3. A variant or fragment of a ligand-dependent corepressor (LCOR) as described in item 1, characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) consists only of a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, or substantially identical thereto.

[0111] 4. The ligand-dependent corepressor (LCOR) described in item 1, characterized in that the above variant or fragment of the ligand-dependent corepressor (LCOR) further comprises the HDAC motif of the amino acid sequence SEQ ID NO: 9, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO: 9. A variant or fragment of )

[0112] 5. A variant or fragment of a ligand-dependent corepressor (LCOR) according to item 4, characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) contains, consists solely of, or substantially consists solely of, a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence SEQ ID NO: 10.

[0113] 6. A variant or fragment of a ligand-dependent corepressor (LCOR) as described in item 4, characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) consists only of sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 12, or substantially identical thereto.

[0114] 7. A variant or fragment of a ligand-dependent corepressor (LCOR) according to item 1 or 4, characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) further comprises either a CTBP1 binding site or / or a CTBP2 binding site.

[0115] 8. A variant or fragment of a ligand-dependent corepressor (LCOR) according to item 1 or 4, characterized in that the above variant or fragment of the ligand-dependent corepressor (LCOR) further comprises a CTBP1 binding site and / or a CTBP2 binding site, wherein the CTBP1 binding site is a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence PLDLTVR (SEQ ID NO: 14) or SEQ ID NO: 14, and the CTBP2 binding site is a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence VLDLSTK (SEQ ID NO: 15) or SEQ ID NO: 15.

[0116] 9. A variant or fragment of a ligand-dependent corepressor (LCOR) according to item 8, characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) consists only of sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 16, or substantially identical thereto.

[0117] 10. A variant or fragment of a ligand-dependent corepressor (LCOR) according to item 8, characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) consists only of, or substantially consists of, a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 18.

[0118] 11. A variant or fragment of a ligand-dependent corepressor (LCOR) as described in item 9, wherein the above variant or fragment of the ligand-dependent corepressor (LCOR) consists solely of SEQ ID NO: 16.

[0119] 12. A variant or fragment of a ligand-dependent corepressor (LCOR) as described in item 9, wherein the above variant or fragment of the ligand-dependent corepressor (LCOR) consists solely of SEQ ID NO: 18.

[0120] 13. Isolated or synthesized variants or fragments of ligand-dependent corepressors (LCORs) described in any one of items 1 to 12, or polynucleotide sequences encoding them, for use as pharmaceuticals.

[0121] 14. Isolated or synthesized mutants or fragments of ligand-dependent corepressors (LCORs) described in any one of items 1 to 12, or polynucleotide sequences encoding them, for use in cancer treatment, preferably for cancer immunotherapy.

[0122] 15. An isolated or synthesized variant or fragment of a ligand-dependent corepressor (LCOR) as described in item 14, or a polynucleotide sequence encoding the same, characterized in that the cancer comprises or consists solely of cancer cells that are positive for androgen, estrogen, or progesterone receptors, and preferably the cancer is estrogen receptor (ER) positive breast cancer.

Claims

1. Isolated or synthesized mutants or fragments of ligand-dependent corepressors (LCORs) for cancer treatment, or polynucleotide sequences encoding such mutants or fragments of ligand-dependent corepressors (LCORs), wherein the mutants or fragments of ligand-dependent corepressors (LCORs) are sequences identical to the amino acid sequence SEQ ID NO: 3, or sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO: 3, and at least one NLS motif (nuclear localization signal) of the amino acid sequence SEQ ID NO:

4. Alternatively, a variant or fragment, or polynucleotide sequence, characterized by containing a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to Sequence ID No. 4, further characterized by the absence or disruption / inactivation of a nuclear receptor-binding domain, wherein the cancer contains or consists solely of cancer cells that are androgen, estrogen, or progesterone receptor-positive, preferably the cancer is estrogen receptor (ER)-positive breast cancer.

2. A variant or fragment of a ligand-dependent corepressor (LCOR) according to claim 1, characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) consists only of a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 5, or substantially identical thereto.

3. A variant or fragment of a ligand-dependent corepressor (LCOR) according to claim 1, characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) consists only of a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, or substantially identical thereto.

4. A variant or fragment of a ligand-dependent corepressor (LCOR) according to claim 1, characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) further comprises the HDAC motif of the amino acid sequence SEQ ID NO: 9, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to SEQ ID NO:

9.

5. A variant or fragment of a ligand-dependent corepressor (LCOR) according to claim 4, characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) contains, consists of, or substantially consists of, a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence SEQ ID NO:

10.

6. A variant or fragment of a ligand-dependent corepressor (LCOR) according to claim 4, characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) consists only of sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence SEQ ID NO: 12, or substantially identical thereto.

7. A variant or fragment of a ligand-dependent corepressor (LCOR) according to claim 1 or 4, characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) further comprises either a CTBP1 binding site or / or a CTBP2 binding site.

8. A variant or fragment of a ligand-dependent corepressor (LCOR) according to claim 1 or 4, characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) further comprises a CTBP1 binding site and / or a CTBP2 binding site, wherein the CTBP1 binding site is a sequence identical to the amino acid sequence PLDLTVR (SEQ ID NO: 14) or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% of SEQ ID NO: 14, and the CTBP2 binding site is a sequence identical to the amino acid sequence VLDLSTK (SEQ ID NO: 15) or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% of SEQ ID NO:

15.

9. A variant or fragment of a ligand-dependent corepressor (LCOR) according to claim 8, characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) consists only of sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence of SEQ ID NO: 16, or substantially identical thereto.

10. A variant or fragment of a ligand-dependent corepressor (LCOR) according to claim 8, characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) consists only of a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence SEQ ID NO: 18, or substantially identical thereto.

11. The ligand-dependent corepressor (LCOR) variant or fragment according to claim 9, wherein the ligand-dependent corepressor (LCOR) variant or fragment comprises SEQ ID NO:

16.

12. The ligand-dependent corepressor (LCOR) variant or fragment according to claim 9, wherein the variant or fragment of the ligand-dependent corepressor (LCOR) consists solely of SEQ ID NO:

18.

13. A variant or fragment of a ligand-dependent corepressor (LCOR) isolated or synthesized, or a polynucleotide sequence encoding such variant or fragment of a ligand-dependent corepressor (LCOR), characterized in that the variant or fragment of the ligand-dependent corepressor (LCOR) consists only of, or substantially only of, a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% identical to the amino acid sequence SEQ ID NO: 16, or SEQ ID NO:

16.

14. The ligand-dependent corepressor (LCOR) variant or fragment according to claim 13, wherein the variant or fragment of the ligand-dependent corepressor (LCOR) consists solely of SEQ ID NO:

16.

15. Isolated or synthesized mutants or fragments of the ligand-dependent corepressor (LCOR) according to claim 13 or 14, or polynucleotide sequences encoding the same, for use as a pharmaceutical agent.

16. Isolated or synthesized mutants or fragments of ligand-dependent corepressors (LCORs) according to claim 13 or 14, or polynucleotide sequences encoding them, for use in cancer treatment, preferably for cancer immunotherapy.

17. The cancer is cancer cells that are positive for androgen, estrogen, or progesterone receptors. An isolated or synthesized variant or fragment of the ligand-dependent corepressor (LCOR) according to claim 16, or a polynucleotide sequence encoding the same, characterized by comprising or consisting solely of, preferably, the cancer being estrogen receptor (ER)-positive breast cancer.

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