FMRP and cancer treatment

Abstract

To provide compositions and methods for the treatment and / or prevention of primary cancer and / or cancer metastasis in a subject in need thereof.SOLUTION: The present invention provides compositions and methods for down-modulating the expression and / or the immuno-suppressive activity of i) the FMRP protein, ii) an mRNA encoding the FMRP protein, and / or iii) the FMR1 gene.SELECTED DRAWING: Figure 1B

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of European Patent Application No. 19172927.6, filed May 7, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present invention provides compositions and methods for modulating the expression and / or activity of i) FMRP protein (hereinafter "FMRP"), ii) mRNA encoding FMRP, and / or iii) the FMR1 gene encoding FMRP for the treatment and / or prevention of cancer and / or cancer metastasis in a subject in need thereof. [Background technology]

[0003] The discovery of immune checkpoint receptors and the development of immunotherapy based on checkpoint blockade have increased the possibility of curing some malignant tumors. 1 This is one of the most notable success stories in basic cancer research and clinical treatment. Immunomodulatory agents targeting T cell co-inhibitory immune checkpoints, such as programmed death-1 (PD-1) or its ligand (PD-L1) and cytotoxic T lymphocyte antigen 4 (CTLA-4), have been approved for the treatment of various types of malignancies. 1 .

[0004] However, across a wide range of human cancer types, a highly variable proportion (40-90%) of patients with various forms of cancer experience little or no benefit from immunotherapy based on known PD-1 or CTLA-4 blockade. 2 This tendency is particularly pronounced in patients with pancreatic ductal adenocarcinoma (PDAC) 3、4 Thus, additional immunotherapeutic strategies remain urgently needed. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Hargadon et al., International Immunopharmacology 62:29-39(2018) [Non-patent document 2] Schoenfeld and Hellman, Cancer Cell 37:443-455 (April 13, 2020) [Non-patent document 3] Royal, RE et al., J. Immunother. 33, 828-833 (2010) [Non-patent document 4] Brahmer, JR et al., New England Journal of Medicine 366, 2455-2465 (2012) Summary of the Invention [Means for solving the problem]

[0006] The present invention provides agents (factors) capable of down-regulating the expression and / or immunosuppressive activity of i) FMRP protein, ii) mRNA encoding FMRP protein, and / or iii) the FMR1 gene encoding FMRP, for use in treating and / or preventing primary cancer and / or cancer metastasis in a subject in need thereof.

[0007] Also provided are plasmids or vectors comprising one or more nucleic acids encoding the miRNA, siRNA, piRNA, hnRNA, snRNA, esiRNA, shRNA, and / or antisense oligonucleotides of the invention.

[0008] Additionally provided are host cells comprising a plasmid or vector of the invention, or one or more nucleic acids encoding the miRNA, siRNA, piRNA, hnRNA, snRNA, esiRNA, shRNA, and / or antisense oligonucleotides of the invention.

[0009] Also provided are plasmids or vectors comprising one or more nucleic acids encoding a peptide or analog thereof, an antibody or antigen-binding fragment thereof, or an antibody mimetic of the invention.

[0010] Additionally, a host cell comprising a plasmid or vector of the invention, or one or more nucleic acids encoding a peptide or analog thereof, an antibody or antigen-binding fragment of the antibody, or an antibody mimetic of the invention is provided.

[0011] Further, there is provided a pharmaceutical composition comprising: i) a therapeutically effective amount of an agent capable of modulating the expression and / or activity of the FMRP protein, the mRNA encoding FMRP, and / or the FMR1 gene; or ii) a plasmid or vector of the invention, or iii) a host cell of the invention; and a pharmaceutically acceptable carrier or diluent; A pharmaceutical composition comprising:

[0012] Additionally provided are pharmaceutical compositions that target FMRP for selective and efficient degradation, comprising an agent disclosed herein, wherein the agent is chemically conjugated to an E3-ubiquitin ligase that tightly binds to FMRP to form an FMRP-agent complex, while the E3-ubiquitin ligase targets the bound protein to the proteasome for degradation.

[0013] Also provided is a method for treating and / or preventing cancer and / or cancer metastasis in a subject in need thereof, comprising the step of administering to the subject an agent of the present invention.

[0014] Also provided is a method for treating and / or preventing cancer and / or cancer metastasis in a subject in need thereof, comprising the step of administering to the subject a pharmaceutical composition of the present invention.

[0015] Without wishing to be bound by any particular theory, it is believed that genetically upregulating the FMR1 gene (endogenously or via gene therapy) in cells or tissues to produce levels of FMRP protein similar to those in many tumors, or delivering FMRP protein or FMR1 mRNA, could be a strategy to ameliorate autoimmune diseases such as type 1 diabetes, which have chronic or other inappropriate infiltration of CD8 (cytotoxic) T cells with associated pathological effects. Similarly, the success of cell therapies involving the transplantation of stem cells and other cells could be enhanced if such cells are engineered to overexpress FMRP, either stably (via lentiviral transduction or CRISPR / Cas9 genome editing) or transiently via AAV. [Brief explanation of the drawings]

[0016] [Figure 1A] Deletion of FMRP in murine PDAC cancer cells significantly extends overall survival and impairs tumor growth and metastasis in immunocompetent, but not immunodeficient, mice. Representative images and quantification of FMRP expression in normal murine pancreas, premalignant PanIN lesions, and PDAC tumor tissue from a P48-cre;LSL-KrasG12D;P53R172H / + PDAC mouse model on an FVBN background. n = 3 mice per group. Student's t-test was used. Scale bar, 100 μm. Immunostaining of human PDAC tissue microarrays (not shown) confirms the results in murine PDAC. [Figure 1B]Deletion of FMRP in murine PDAC cancer cells significantly extends overall survival and impairs tumor growth and metastasis in immunocompetent, but not immunodeficient, mice. Expression of FMRP in a "wild-type" (WT) murine PDAC cell line (4361.12) and non-expression of FMRP in derivative FMRP-deficient cells ("KO") generated by transient transfection with a Cas9 / sgRNA vector targeting the murine FMR1 gene encoding FMRP were demonstrated by Western blotting using two FMRP antibodies (Abcam, ab191411; Cell Signaling, 4317s) that recognize different epitopes on the FMRP protein. Three independent experiments were performed. [Figure 1C] Deletion of FMRP in murine PDAC cancer cells significantly extends overall survival and impairs tumor growth and metastasis in immunocompetent, but not immunodeficient, mice. Colony formation of cultured 4361.12 WT2 and FMRP KO2 cells. The indicated number of cancer cells was seeded in a single well of a 6-well plate. After 10 days, cells were fixed and stained with crystal violet. Three independent experiments. [Figure 1D] Deletion of FMRP in murine PDAC cancer cells significantly prolongs overall survival and impairs tumor growth and metastasis in immunocompetent, but not immunodeficient, mice. Schematic of the in vivo lung metastasis assay. Briefly, 2 x 10 cells were injected into the tail vein of immunocompetent FVBN or immunodeficient SCID / Beige mice, allowing cancer cells to seed the lungs. Mice were monitored twice weekly and sacrificed upon reaching veterinary endpoints. [Figure 1E] Deletion of FMRP in murine PDAC cancer cells significantly extends overall survival and impairs tumor growth and metastasis in immunocompetent, but not immunodeficient, mice. Overall survival of syngenic immunocompetent FVBn mice injected with murine PDAC WT2 or FMRP KO2 cells, n=5 mice per group, using Kaplan-Meier testing. [Figure 1F] Deletion of FMRP in murine PDAC cancer cells significantly extends overall survival and impairs tumor growth and metastasis in immunocompetent, but not immunodeficient, mice. Overall survival of syngenic immunodeficient SCID / Beige mice injected with murine PDAC WT2 or FMRP KO2 cells, n=5 mice per group, Kaplan-Meier test. [Figure 1G] Deletion of FMRP in murine PDAC cancer cells significantly extends overall survival and impairs tumor growth and metastasis in immunocompetent, but not immunodeficient, mice. Western blot analysis revealed FMRP expression in WT cells of the murine PDAC cell line 4361.12 and non-expression in a second FMRP KO cell line (KO8), generated by transient transfection with a Cas9 / SgRNA vector also targeting the murine FMR1 gene. Three independent experiments. [Figure 1H] Deletion of FMRP in murine PDAC cancer cells significantly prolongs overall survival and impairs tumor growth and metastasis in immunocompetent, but not immunodeficient, mice. Schematic of the in vivo subcutaneous (sc) primary tumor growth model. Briefly, 5 x 10 cells were injected subcutaneously into FVBN or NSG mice. Tumor-bearing mice were monitored twice weekly and sacrificed 25 days post-injection when WT tumor volumes reached 1000 mm3. [Figure 1I] Deletion of FMRP in murine PDAC cancer cells significantly prolongs overall survival and impairs tumor growth and metastasis in immunocompetent, but not immunodeficient, mice. Tumor weights at day 28 in FVBn(I) mice injected with murine PDAC WT (two independent clones WT2 and WT3) or FMRP KO (two independent clones KO2 and KO8) cells were calculated using an unpaired t-test. [Figure 1J]Deletion of FMRP in murine PDAC cancer cells significantly prolongs overall survival and impairs tumor growth and metastasis in immunocompetent, but not immunodeficient, mice. Tumor weights at day 28 in immunodeficient NSG mice injected with murine PDAC WT (two independent clones WT2 and WT3) or FMRP KO (two independent clones KO2 and KO8) cells were calculated using an unpaired t-test. [Figure 2A] Deletion of FMRP in mouse PDAC cancer cells induces a strong antitumor immune response in the form of infiltrating CD8+ (cytotoxic) T lymphocytes. Immunochemical staining and quantification of CD8+ (cytotoxic) T lymphocytes in primary tumors formed by mouse PDAC WT and KO cells. Scale bar, 100 μm. n = 3 mice per group. [Figure 2B] Deletion of FMRP in mouse PDAC cancer cells induces a strong antitumor immune response in the form of infiltrating CD8+ (cytotoxic) T lymphocytes. IF staining and quantification of CD45+ immune cells in primary tumors formed by mouse PDAC WT2 and KO2 cells. Scale bar, 100 μm. n = 3 mice per group. [Figure 2C] Deletion of FMRP in murine PDAC cancer cells elicits a strong antitumor immune response in the form of infiltrating CD8+ (cytotoxic) T lymphocytes. FACS analysis was used to measure the frequency of CD45+ immune cells in PDAC WT and FMRP KO tumors grown in immunocompetent mice. n = 4–5 mice per group, two independent experiments. Unpaired t-tests were used. [Figure 2D] Deletion of FMRP in murine PDAC cancer cells elicits a strong antitumor immune response in the form of infiltrating CD8+ (cytotoxic) T lymphocytes. Using FACS analysis, we measured the frequency of CD3+CD8+ and CD8+ T cells in PDAC WT and FMRP KO tumors grown in immunocompetent mice. n = 4–5 mice per group, two independent experiments. Unpaired t-tests were used. [Figure 2E]Deletion of FMRP in murine PDAC cancer cells elicits a strong antitumor immune response in the form of infiltrating CD8+ (cytotoxic) T lymphocytes. FACS analysis was used to measure the frequency of activated GRZb+ cells in PDAC WT and FMRP KO tumors grown in immunocompetent mice. n = 4–5 mice per group, two independent experiments. Unpaired t-tests were used. [Figure 2F] Deletion of FMRP in murine PDAC cancer cells elicits a strong antitumor immune response in the form of infiltrating CD8+ (cytotoxic) T lymphocytes. FACS analysis was used to measure the frequency of activated IFNγ+ in PDAC WT and FMRP KO tumors grown in immunocompetent mice. n = 4–5 mice per group, two independent experiments. Unpaired t-tests were used. [Figure 2G] Deletion of FMRP in murine PDAC cancer cells elicits a strong antitumor immune response in the form of infiltrating CD8+ (cytotoxic) T lymphocytes. FACS analysis was used to measure the frequency of activated TNFα+ cells in PDAC WT and FMRP KO tumors grown in immunocompetent mice. n = 4–5 mice per group, two independent experiments. Unpaired t-tests were used. [Figure 2H] Deletion of FMRP in mouse PDAC cancer cells elicits a strong antitumor immune response in the form of infiltrating CD8+ (cytotoxic) T lymphocytes. Representative double immunostaining images show FMRP and CD8 expression in the center and margins of mouse PDAC tissue from a P48-cre;LSL-KrasG12D;P53R172H / + PDAC mouse model on an FVBN background. n = 8 mouse PDAC samples. Paired t-test was used. Scale bar, 100 μm. The data support the interpretation that FMRP prevents the influx of CD8 T cells seen in KO tumors (Panel A). Immunostaining of human PDAC tissue microarrays for CD8 and FMRP (not shown) demonstrated an inverse correlation between the density of infiltrating CD8 T cells and FMRP expression, consistent with the results in mouse PDAC. [Figure 3A]FMRP KO in cancer cells sensitizes otherwise resistant PDAC tumors to immunotherapy, including anti-PD1 antibody treatment. FMRP deletion does not suppress PD-L1 expression in mouse PDAC cells in vitro or in vivo. WB analysis of PD-L1 expression in mouse PDAC WT and FMRP KO cells, from three independent experiments. [Figure 3B] FMRP KO in cancer cells sensitizes otherwise resistant PDAC tumors to immunotherapy, including anti-PD1 antibody treatment. FMRP deletion does not suppress PD-L1 expression in mouse PDAC cells in vitro or in vivo. Immunostaining to detect PD-L1 expression in tumors formed by mouse PDAC WT and FMRP KO cells. Scale bar, 100 μm. n = 3 mice per group. [Figure 3C] FMRP knockout in cancer cells sensitizes otherwise resistant PDAC tumors to immunotherapy, including anti-PD-1 antibody treatment. Growth curves of PDAC WT2 and KO2 tumors in FVBn mice without and with anti-PD-1 antibody treatment (IP, 200 μg per mouse, twice weekly). n = 7–11 mice per group, unpaired t-test. The results indicate that PDAC tumors arising from this murine PDAC cancer cell line are less sensitive to anti-PD-1 therapy. [Figure 3D] FMRP KO in cancer cells sensitizes otherwise resistant PDAC tumors to immunotherapy, including anti-PD1 antibody treatment. Growth curves of PDAC WT2 and KO2 tumors in FVBn mice without and with anti-PD-1 antibody treatment (IP, 200 μg per mouse, twice weekly) were shown. n = 7–11 mice per group, unpaired t-test was used. In stark contrast, FMRP KO significantly impaired PDAC tumor growth, associated with increased CD8 T cell influx (as shown in Figures 1G–I, 2A, and 4D). This suggests that FMRP inhibitors may have therapeutic efficacy in tumors resistant to anti-PD1 / PD-L1 therapy. [Figure 4A]Combined deletion of the gene encoding FMRP and the gene encoding the RNA A to I-editing protein ADAR1 in mouse PDAC further extended survival. FMRP and ADAR1 interact in PDAC cancer cells, as revealed by co-immunoprecipitation experiments in mouse PDAC 4361.12 WT2 cells. FMRP and ADAR1 were visualized by Western blot of whole cell lysates before and after immunoprecipitation with rabbit anti-FMRP antibody (Abcam, ab191411). Normal rIgG antibody was used as a control. Three independent experiments. [Figure 4B] Combined deletion of the gene encoding FMRP and the gene encoding the RNA A to I-editing protein ADAR1 in mouse PDAC further extended survival. FMRP-ADAR1 interaction was also verified by reverse co-immunoprecipitation experiments. FMRP and ADAR1 were revealed by immunostaining Western blots of whole-cell lysates before and after immunoprecipitation with mouse anti-ADAR1 antibody (Santa Cruz, sc73408). Normal mIgG antibody was used as a control. Three independent experiments. [Figure 4C] Combined deletion of the gene encoding FMRP and the gene encoding the RNA A to I-editing protein ADAR1 in mouse PDAC further extended survival. FMRP and ADAR1 expression was examined by Western blot analysis in WT2, FMRP KO, ADAR1 KO, and FMRP / ADAR1 double KO cells. These cells were generated by transient transfection with Cas9 / sgRNA vectors targeting the mouse FMR1 and ADAR1 genes. This was performed in three independent experiments. [Figure 4D]Combined deletion of the gene encoding FMRP and the gene encoding the RNA A to I-editing protein ADAR1 in murine PDAC further extended survival. Overall survival of FVBn mice injected with WT2, FMRP KO, ADAR1 KO, and FMRP / ADAR1 double KO cells was measured using the Kaplan-Meier test. Briefly, 5 x 10 cells were injected sc into the flank of FVBN mice. Mice were monitored twice weekly and sacrificed when tumor volume reached 1000 mm3. [Figure 5A] Deletion of FMRP in cancer cells of a second tumor type, colon carcinoma, similarly impairs tumor growth in immunocompetent, but not immunodeficient, mice. Representative images and quantification of immunohistochemical staining of FMRP expression in normal colon and adenoma tissue from mice in the AKP (ApcΔ / Δ; KrasG12D / +; Trp53Δ / Δ; CDX2 Cre ERT2) or APC (ApcΔ / Δ; CDX2 Cre ERT2) mouse models. n = 3 mice per group. Student's t-test was used. Scale bar, 100 μm. Immunostaining of human colon cancer tissue microarrays (not shown) confirms the mouse data. [Figure 5B] Deletion of FMRP in colon cancer cells similarly impairs tumor growth in immunocompetent, but not immunodeficient, mice. FMRP expression was verified by Western blot analysis in CT26 WT and FMRP KO subclones generated by transient transfection with a Cas9 / SgRNA vector targeting the mouse FMR1 gene. FMRP deletion in CT26 cells was verified using two antibodies (Abcam, ab191411; Cell Signaling Technology, CST, #4317) that recognize different epitopes on the FMRP protein. Three independent experiments were performed. [Figure 5C]Deletion of FMRP in cancer cells of a second tumor type, colon carcinoma, similarly impairs tumor growth in immunocompetent but not immunodeficient mice. Colony formation assay of WT17# and KO12# cells from CT26. 1250 cancer cells were seeded per well of a 6-well plate. After 10 days, cells were fixed and stained with crystal violet. Three independent experiments. [Figure 5D] Deletion of FMRP in cancer cells of a second tumor type, colon carcinoma, similarly impairs tumor growth in immunocompetent but not immunodeficient mice. Schematic of the in vivo subcutaneous (sc) primary tumor growth model. Briefly, 5 x 10 cells were injected sc into immunocompetent Balb / c mice or immunodeficient NSG mice. Mice were monitored twice weekly and sacrificed on days 25 and 18 post-injection, respectively, when WT tumor volumes reached 1000 mm3. [Figure 5E] Deletion of FMRP in cancer cells of a second tumor type, colon carcinoma, similarly impairs tumor growth in immunocompetent, but not immunodeficient, mice. Tumor growth curves of Balb / c mice injected with CT26 WT17# or FMRP KO12# cells up to 25 days after sc injection, n = 10 mice per group, unpaired t-test. [Figure 5F] Deletion of FMRP in cancer cells of a second tumor type, colon carcinoma, similarly impairs tumor growth in immunocompetent but not immunodeficient mice. Representative images and tumor weights at day 25 in Balb / c mice injected with CT26 WT17# or FMRP KO12# cells, n = 10 mice per group, unpaired t-test. [Figure 5G]Deletion of FMRP in cancer cells of a second tumor type, colon carcinoma, similarly impairs tumor growth in immunocompetent but not immunodeficient mice. Immunochemical staining of CD8 and FMRP in primary tumors formed by CT26 WT17# or FMRP KO12# cells, scale bar, 100 μm, n = 3 mice per group (left panel); quantification of CD8+ T cells in primary tumors formed by CT26 WT17# or FMRP KO12# cells (right panel); n = 3 mice per group. Student's t-test was used. Scale bar, 100 μm. [Figure 5H] Deletion of FMRP in cancer cells of a second tumor type, colon carcinoma, similarly impairs tumor growth in immunocompetent but not immunodeficient mice. Tumor growth curves for NSG mice injected sc with CT26 WT12# or FMRP KO12# cells up to 14 days post-injection, n = 5 mice per group, unpaired t-test. [Figure 5I] Deletion of FMRP in cancer cells of a second tumor type, colon carcinoma, similarly impairs tumor growth in immunocompetent, but not immunodeficient, mice. Representative images and tumor weights at day 14 in NSG mice injected with CT26 WT17# or FMRP KO12# cells, n = 5 mice per group, unpaired t-test. [Figure 6A] Deletion of FMRP in mouse melanoma cells significantly impairs tumor growth in immunocompetent mice. Representative images and quantification of immunohistochemical staining of FMRP expression in normal skin and melanoma tissues of mice from the iBIP2 (inducible BRAF INK / ARF PTEN) melanoma mouse model on an FVBN background. Normal skin group: n = 2 mice, iBIP2 melanoma group: n = 4 mice. Student's t-test was used. Scale bar: 100 μm. [Figure 6B]Deletion of FMRP in mouse melanoma cells significantly impairs tumor growth in immunocompetent mice. FMRP expression was verified by Western blot analysis in B16-OVA WT and FMRP KO subclones generated by transient transfection with a Cas9 / SgRNA vector targeting the mouse FMR1 gene. Three independent experiments. [Figure 6C] Deletion of FMRP in mouse melanoma cells significantly impairs tumor growth in immunocompetent mice. Colony formation assay of B16-OVA WT and FMRP KO cells. 1250 cancer cells were seeded in one well of a 6-well plate. After 10 days, cells were fixed and stained with crystal violet. Three independent experiments. [Figure 6D] Deletion of FMRP in mouse melanoma cells significantly impaired tumor growth in immunocompetent mice. Tumor growth curves were obtained from C57B / 6 mice injected sc with B16-OVA WT or FMRP KO cells up to day 18 post-injection (n = 5-10 mice per group, unpaired t-test). Briefly, 5 x 10 cells were injected sc into the flank of C57B / 6 mice. Mice were monitored twice weekly and sacrificed on day 18 post-injection when WT tumor volumes reached 1000 mm3. [Figure 6E] Deletion of FMRP in mouse melanoma cells significantly impairs tumor growth in immunocompetent mice. Representative images and tumor weights of immunocompetent mice injected with B16-OVA WT or FMRP KO cells, collected on day 18, n = 5–10 mice per group. Unpaired t-test was used. [Figure 7A] In the genetically engineered RIP1-Tag2 (RT2) mouse model of multistage pancreatic neuroendocrine neoplasia (PanNET), specific deletion of FMRP in early cancer cells significantly extends survival. Representative images of immunohistochemical staining for FMRP expression in normal mouse pancreas, PanNET tumors, and liver metastases. n = 3 mice per group. Scale bar, 100 μm. [Figure 7B]In the genetically engineered RIP1-Tag2 (RT2) mouse model of multistage pancreatic neuroendocrine tumorigenesis (PanNET), specific deletion of FMRP in early-stage cancer cells significantly extended survival. Overall survival times for male RT2 and FMRP KO RT2 mice were measured using the Kaplan-Meier test. Male FMRP KO RT2 mice, in which the FMR1 gene encoding FMRP was specifically deleted in pancreatic islet β cells expressing the SV40 oncogene that promotes PanNET tumorigenesis, were generated by crossing FMR1 floxed mice with Rip1-Tag2 (RT2) and RIP7-Cre mice. The FMRP KO RT2 group consisted of n=17 and the RT2 group consisted of n=12. All mice were monitored twice weekly and sacrificed upon reaching veterinary endpoints. [Figure 8A] Elevated FMRP expression in mouse breast cancer tissue. Representative images and quantification of FMRP expression in normal mouse mammary fat pads (MFPs) and de novo breast tumors in the genetically engineered MMTV-PymT breast cancer mouse model. n = 3 mice per group. Student's T-test was used. Scale bar, 100 μm. [Figure 8B] Elevated FMRP expression in mouse breast cancer tissue. Representative images and quantification of FMRP expression in normal mouse mammary fat pads (MFPs) and de novo breast tumors from a genetically engineered C3Tag triple-negative breast cancer (TNBC) mouse model. n = 3 mice for the normal MFP group and n = 3 mice for the C3Tag breast cancer group. Student's t-test was used. Scale bar, 100 μm. Immunostaining of human triple-negative breast cancer (TNBC) tissue microarrays (not shown) confirms the results in mouse breast cancer. [Figure 9A]Inhibition of FMRP by siRNA in mouse PDAC cells. Migration assay of mouse PDAC 4361.12 WT2 and FMRP KO2 cells. Briefly, 5000 cells were seeded in 50 μl of serum-free DMEM medium into the upper well of a Boyden chamber (membrane pore size, 8 μm), and 200 μl of DMEM medium containing FBS was placed in the lower chamber. After 18 hours, remaining cancer cells in the well were removed using a cotton swab soaked in 70% EtOH. Cells that migrated through the 8 μm pores on the membrane were fixed and then stained with crystal violet. The number of migrated cells was counted. Data were collected from three independent wells. An unpaired t-test was used. Three independent experiments were performed. [Figure 9B] Inhibition of FMRP by siRNA in mouse PDAC cells. FMRP expression was examined by Western blot in mouse PDAC 4361.12 WT2 cells transfected with control siRNA (i.e., siCtrl: UAAGG CUAUG AAGAG AUAC (SEQ ID NO: 9)) and FMRP-targeting siRNA (siFMRP#1: AUAAG AGACA ACUUG GUGC (SEQ ID NO: 10) and siFMRP#2: UAACUUCGGAAUUAUGUAG (SEQ ID NO: 11)). Three independent experiments were performed. [Figure 9C] Inhibition of FMRP by siRNA in mouse PDAC cells. Migration assay of mouse PDAC 4361.12 WT2 cells transfected with control siRNA and FMRP-targeting siRNA; 5000 cells per well, 18 hours. Data collected from three independent wells. Unpaired t-test was used. Three independent experiments. [Figure 10A]FRET-based high-throughput screening (HTS) for FMRP inhibitors. Schematic diagram of FRET-based high-throughput screening (HTS) for FMRP inhibitors. Human FMRP protein is produced in human HEK293 cells, purified, and biochemically labeled with the fluorescent reporter fluorescein. 2) sc1 RNA1 is labeled with a fluorescent quencher molecule, Cy3 or BHQ, so that the excitable fluorescence emission of FITC is quenched when sc1 binds to FMRP. 3) Compounds that elicit the emission of quenched fluorescence are further characterized to verify their ability to inhibit the interaction between FMRP and sc1. [Figure 10B] High-throughput screening (HTS) of FRET-based FMRP inhibitors. Purification profile of mammalian-expressed FMRP-His protein. Human FMRP protein is produced in human HEK293 cells, purified, and validated. The left panel shows Coomassie blue staining showing 2 μg of total protein in each lane. The right panel shows Western blot of FMRP-His protein expression using an anti-His Tag antibody. M: Molecular weight marker. Me: Culture medium. FT: Flow-through. W: Wash. E: Eluted fraction. The eluted fractions were pooled, buffer exchanged, and concentrated.

[0017] Figure 11 shows that FMRP is widely and highly expressed in various types of human cancer (adapted from The Human Protein Atlas; https: / / www.proteinatlas.org / ENSG00000102081-FMR1 / pathology). DETAILED DESCRIPTION OF THE INVENTION

[0018] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Additionally, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0019] In case of conflict, the present specification, including definitions, will control. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs. As used herein, the following definitions are provided to facilitate understanding of the present invention.

[0020] The term "comprise / comprising" is generally used in the sense of include / including, i.e. allowing for the presence of one or more features or components.

[0021] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0022] As used herein, "at least one (one type)" means "one (one type) or more," "two (two types) or more," "three (three types) or more," etc.

[0023] As used herein, the terms "subject" / "subject in need" or "patient" / "patient in need" are well-recognized in the art and are used interchangeably herein to refer to mammals, including dogs, cats, rats, mice, monkeys, cows, horses, goats, sheep, pigs, camels, and most preferably humans. In some cases, the subject is a subject in need of treatment or a subject suffering from a disease or disorder. However, in other embodiments, the subject can be a healthy subject. The term does not denote a particular age or sex. Thus, it is intended to encompass adult and newborn subjects, whether male or female. Preferably, the subject is a human. Most preferably, the subject is a human suffering from cancer and / or cancer metastasis or who may be at risk of suffering from cancer and / or cancer metastasis.

[0024] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to any type of polymer of deoxyribonucleotides (e.g., DNA, cDNA, ...) or ribonucleotides (e.g., RNA, mRNA, ...), or a combination of deoxyribonucleotide and ribonucleotide polymers (e.g., DNA / RNA), in linear or circular conformation, in single- or double-stranded form. These terms are not intended to be restrictive with respect to the length of the polymer and can encompass known analogues of natural nucleotides, as well as nucleotides modified in the base, sugar, and / or phosphate moieties (e.g., phosphorothioate backbones). In general, analogues of a particular nucleotide have the same base-pairing specificity; i.e., an analogue of A will base-pair with T.

[0025] The term "vector," as used herein, refers to a nucleic acid (DNA or RNA) molecule, such as a viral vector, or a plasmid or other vehicle, which contains one or more heterologous nucleic acid sequences of the invention and is preferably designed for transfer (transduction) between different host cells. The terms "expression vector," "gene transfer vector," and "gene therapy vector" refer to any vector effective for incorporating and expressing one or more nucleic acids of the invention in a cell, preferably under the control of a promoter. A cloning vector or expression vector may contain additional elements in addition to a promoter, such as, for example, regulatory elements and / or post-transcriptional regulatory elements.

[0026] The term "about," particularly with respect to a given quantity, is meant to encompass a deviation of ±10%.

[0027] The present inventors have focused on the role of FMRP in promoting the invasive development of pancreatic neuroendocrine carcinoma and ductal carcinoma. 5 , unexpectedly discovered an unexpected and unprecedented role for FMRP in suppressing antitumor immunity in vivo.

[0028] Fragile X mental retardation protein (FMRP) is an RNA-binding protein highly expressed in the brain that binds to a specific subset of mRNAs for synaptic (and other) proteins and regulates their translation within neurons. 6 FMRP plays a critical role in synaptic function, and its deficiency leads to fragile X syndrome (FXS), the most common form of inherited intellectual disability and one of the leading causes of autism. 7 Contrary to this role, other studies of FMRP have revealed its expression in several types of cancer. 8、9 , which has been implicated in cancer cell survival, invasion, and metastasis. In this disclosure, the term FMRP also refers to FMRP isoforms.

[0029] We have shown that lack of FMRP expression in murine pancreatic ductal adenocarcinoma (PDAC) and colon cancer cells significantly prolongs overall survival and impairs tumor growth in syngenic, immunocompetent mice.

[0030] The present invention provides agents capable of modulating the expression and / or activity of i) FMRP protein, ii) mRNA encoding FMRP, and / or iii) FMR1 gene, for use in the treatment and / or prevention of cancer and / or cancer metastasis in a subject in need thereof.

[0031] Preferably, modulation of FMRP protein expression and / or activity involves regulatory interaction of the FMRP protein with its mRNA and miRNA targets (e.g., via its RNA-binding domain) and / or regulatory interaction with other proteins.

[0032] In certain embodiments, the modulation is a decrease in FMRP mRNA levels. In certain embodiments, the modulation is a decrease in FMRP protein levels and / or activity. In certain embodiments, the levels of both FMRP mRNA and FMRP protein are decreased. Such a decrease may occur in a time-dependent or dose-dependent manner.

[0033] As used herein, "inhibition" or "reduction" or "reduction" are used interchangeably to mean a decrease in target nucleic acid levels or target protein levels in the presence of an agent of the invention compared to the target nucleic acid levels or target protein levels in the absence of the agent of the invention.

[0034] In one embodiment, an agent of the invention inhibits translation of RNA encoding FMRP.

[0035] In another embodiment, an agent of the invention inhibits transcription of DNA encoding FMRP.

[0036] In a further aspect, the agent inhibits or impairs binding of FMRP to a target mRNA and / or the agent inhibits or impairs binding of FMRP to an interacting protein or other molecule through which FMRP mediates its immunosuppressive activity.

[0037] Preferably, the cancer and / or cancer metastasis to be treated is resistant to immunotherapy and is selected from non-limiting examples of cancers including carcinoma, blastoma, sarcoma, melanoma, lymphoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include breast cancer, colon cancer, rectal cancer, colorectal cancer, kidney cancer or renal cancer, small cell lung cancer, non-small cell lung cancer, clear cell carcinoma including adenocarcinoma of the lung and squamous cell carcinoma of the lung, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), cervical cancer, ovarian cancer, prostate cancer, prostate neoplasms, liver cancer, bladder cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer including gastric cancer and stomach cancer. cancer), gastrointestinal stromal tumor, pancreatic cancer, head and neck cancer, glioblastoma, retinoblastoma, astrocytoma, theca cell tumor, androgenic tumor, hepatoma, non-Hodgkin's lymphoma (NHL), multiple myeloma, myelodysplastic disorder, myeloproliferative disorder, chronic myeloid leukemia, hematologic malignancies including acute hematologic malignancies, endometrial or uterine cancer, endometriosis, endometrial stromal sarcoma, fibrosarcoma, choriocarcinoma, salivary gland cancer, vulvar cancer, thyroid cancer, esophageal cancer, hepatocellular carcinoma, anal cancer, genital cancer These include stalk cancer, nasopharyngeal cancer, laryngeal cancer, Kaposi's sarcoma, mast cell sarcoma, ovarian sarcoma, uterine sarcoma, melanoma, malignant mesothelioma, skin cancer, schwannoma, oligodendroglioma, neuroblastoma, neuroectodermal tumor, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcoma, Ewing's sarcoma, peripheral primitive neuroectodermal tumor, urinary tract cancer, thyroid cell carcinoma, Wilms' tumor, as well as abnormal vascular proliferation associated with nevus syndrome, edema (such as edema associated with brain tumors), and Meigs' syndrome.

[0038] Typically, the agent of the invention is a chemical compound, a peptide or analog thereof, a nucleic acid, an antibody, an antigen-binding fragment of the antibody, or an antibody mimetic. Preferably, the agent is capable of accessing the intracellular compartment of cancer cells, given the predominant intracellular localization of FMRP in the cytoplasm.

[0039] As used herein, a "chemical agent or compound" refers to a compound that produces changes through its chemical composition and its effect on living tissues and organisms. A chemical agent may be a small molecule inhibitor (SMI), a nucleic acid, e.g., siRNA, or a peptide. In embodiments, the compound is preferably a non-peptidyl molecule. Most preferably, the non-peptidyl molecule induces selective intracellular proteolysis of a peptide encoded by a nucleic acid sequence of the present invention. Examples of compounds that induce selective intracellular proteolysis include proteolysis-inducing chimeric molecule (PROTAC) proteolytic agents and small molecule chemical modulators (factors) of deubiquitinases upstream or downstream of the proteasome. As known in the art, PROTACs (also known as active degraders) are heterobifunctional small molecules consisting of two active domains and a linker that can remove specific unwanted proteins.

[0040] Compositions of matter that inhibit FMRP and thereby mimic the invention revealed by gene knockout can be discovered and validated using many techniques and methodologies known to those skilled in the art. The following are illustrative of the spectrum of approaches that can be used to identify FMRP inhibitors with potential for therapeutic development as anti-cancer agents.

[0041] If the agent is a peptide, the agent is preferably conjugated to an agent that increases the accumulation of the peptide in cancer cells. Such agents can be, for example, compounds that induce receptor-mediated endocytosis, such as membrane transferrin receptor-mediated endocytosis of transferrin conjugated with a therapeutic agent (Qian ZM et al., "Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway", Pharmacological Reviews, 54, 561, 2002), or cell membrane-permeable carriers that can be selected from the group of fatty acids, such as decanoic acid, myristic acid, and stearic acid, which inhibit protein kinase C (Ioannides CG et al., "Inhibition of IL-2 receptor induction and IL-2 production in the human leukemic cell line Jurkat by a novel peptide inhibitor of protein kinase C", Cell Immunol, 131, 242, 1990) and protein tyrosine phosphatase (Kole HK et al., "A peptide-based protein-tyrosine phosphatase inhibitor specifically enhances insulin receptor function in intact The carrier may be a cell membrane-permeable carrier that has already been used for intracellular delivery of peptide inhibitors ("Inhibition of Inhibitory Effects on Cells," J. Biol. Chem., 271, 14302, 1996), or may be selected from peptides. Preferably, a cell membrane-permeable carrier is used. More preferably, a cell membrane-permeable carrier peptide is used.

[0042] When the cell membrane permeable carrier is a peptide, it is preferably a positively charged amino acid-rich peptide.

[0043] Preferably, such a positively charged amino acid-rich peptide is an arginine-rich peptide. Futaki et al. (Futaki S. et al., "Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery", J. Biol. Chem., 276, 5836, 2001) have shown that the number of arginine residues in a cell membrane-permeable carrier peptide has a significant effect on the method of internalization, and that there appears to be an optimal number of arginine residues for internalization, preferably containing more than 6 arginines, more preferably containing 9 arginines (R9).

[0044] The peptide may be linked to the cell membrane permeable carrier via a spacer (e.g., two glycine residues). Any cell membrane permeable carrier can be used as determined by those skilled in the art. In this case, the cell membrane permeable carrier is preferably a peptide.

[0045] Typically, the arginine-rich peptide is selected from the non-limiting group including HIV-TAT 48-57 peptide (GRKKRRQRRR; SEQ ID NO: 14), FHV-coat 35-49 peptide (RRRRNRTRRNRRRVR; SEQ ID NO: 15), HTLV-II Rex 4-16 peptide (TRRQRTRRARRNR; SEQ ID NO: 16), and BMV gag 7-25 peptide (KMTRAQRRAAARRNRWTAR) (SEQ ID NO: 17).

[0046] Since an inherent problem with unmodified (native) peptides (L-form) is degradation by natural proteases, the peptides of the present invention and cell membrane-permeable peptides may be prepared to contain D-forms and / or "retro-inverso isomers" of the peptides. In this case, retro-inverso isomers of fragments and variants of the peptides of the present invention and cell membrane-permeable peptides are prepared.

[0047] When the agent is a nucleic acid, the agent is selected from the group including nucleic acids encoding miRNA, siRNA, piRNA, hnRNA, snRNA, sgRNA, esiRNA, shRNA, and antisense oligonucleotides (e.g., modified ASOs), or combinations thereof.

[0048] When the agent is a nucleic acid, the agent can be prepared by any suitable art-recognized method, such as phosphoramidite chemistry or H-phosphonate chemistry, which can be performed manually or by an automated synthesizer. The nucleic acid-based agents of the present invention may be modified in many ways without impairing their ability to hybridize to their targets (see, e.g., Agrawal and Gait, Advances in Nucleic Acid Therapeutics, (2019) https: / / doi.org / 10.1039 / 9781788015714).

[0049] In embodiments in which the agent is an miRNA, siRNA, piRNA, hnRNA, snRNA, sgRNA, esiRNA, shRNA, or antisense compound, the agent is targeted to a human FMRP nucleic acid. Nucleotide sequences encoding human FMRP include, but are not limited to, GENBANK Accession No. NM_001185075.1 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NM_001185076.1 (incorporated herein as SEQ ID NO: 2), GENBANK Accession No. NM_001185081.2 (incorporated herein as SEQ ID NO: 3), GENBANK Accession No. NM_001185082.2 (incorporated herein as SEQ ID NO: 4), and GENBANK Accession No. NM_002024.6 (incorporated herein as SEQ ID NO: 5).

[0050] Nucleotide sequences encoding mouse FMRP include, but are not limited to, GENBANK Accession No. NM_001290424.1 (incorporated herein as SEQ ID NO:6), GENBANK Accession No. NM_001374719.1 (incorporated herein as SEQ ID NO:7), and GENBANK Accession No. NM_008031.3 (incorporated herein as SEQ ID NO:8).

[0051] The terms "microRNA," "miRNA," and "MiR" are used interchangeably and refer to endogenous or artificial non-coding RNAs that can regulate gene expression. miRNAs are believed to function through RNA interference. Designing such microRNAs is within the skill of one of ordinary skill in the art.

[0052] The terms "siRNA" and "small interfering RNA" are used interchangeably and refer to single- or double-stranded RNA molecules capable of inducing RNA interference. siRNA molecules typically have a double-stranded region 18-30 base pairs in length. Design of such siRNAs is within the skill of those in the art.

[0053] The terms "piRNA" and "Piwi-binding RNA" are used interchangeably and refer to a class of small RNAs involved in gene silencing. piRNA molecules are typically 26-31 nucleotides in length. Designing such PiRNAs is within the skill of one of ordinary skill in the art.

[0054] An example of a modified antisense oligonucleotide (ASO) is a GapmeR. As used herein, a GapmeR is a chimeric antisense oligonucleotide containing a central block of deoxynucleotide monomers of sufficient length to induce RNase H cleavage. Typically, the GapmeR of the present invention is directed against one or more mRNAs encoding FMRP or target mRNAs. The design of such GapmeRs is within the skill of one of ordinary skill in the art.

[0055] The terms "sgRNA" and "guide RNA" are used interchangeably to refer to specific RNA sequences that recognize a target DNA region of interest and guide an endonuclease there for editing. gRNAs typically consist of two parts: crisprRNA (crRNA), a 17-20 nucleotide sequence complementary to the target DNA, and tracrRNA, which serves as a binding scaffold for Cas nucleases.

[0056] Any suitable engineered sgRNA, crRNA, or tracrRNA can be employed as long as it is effective in recognizing the target DNA of the present invention. Design of such sgRNA, crRNA, and tracrRNA is within the skill of those skilled in the art. For example, the sgRNA can be directed to recognize the DNA of FMR1, and can be, for example, an sgRNA selected from the group including 5'-GTGGAAGTGCGGGGCTCCAA-3' (SEQ ID NO: 12) and 5'-GAGCTGGTGGTGGAAGTGCG-3 (SEQ ID NO: 13), or a combination thereof.

[0057] The terms "snRNA" and "small nuclear RNA" are used interchangeably to refer to a class of small RNAs involved in various processes, including RNA splicing and regulation of transcription factors. Also included are subclasses of small nucleolar RNAs (snoRNAs). The term is also intended to include artificial snRNAs, such as antisense derivatives of snRNAs. Design of such snRNAs is within the skill of one in the art.

[0058] Therefore, in particular, the present invention provides isolated siRNAs comprising short double-stranded RNAs of about 18 to about 30 nucleotides in length that target mRNA encoding FMRP or a target mRNA. The term "isolated" means altered or removed from its natural state by human intervention. For example, siRNAs naturally occurring in a living animal are not "isolated," whereas synthetic siRNAs and siRNAs partially or completely separated from coexisting materials in their natural state are "isolated." Isolated siRNAs can exist in a substantially purified form or can exist in a non-native environment, such as a cell into which the siRNA is delivered. The siRNAs of the present invention can include partially purified RNA, substantially pure RNA, synthetic RNA, or recombinantly produced RNA, as well as modified RNAs that differ from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such modifications can include the addition of non-nucleotide material, such as to one or both termini of the siRNA or to one or more internal nucleotides of the siRNA, including modifications that render the siRNA resistant to nuclease digestion.

[0059] One or both strands of the siRNA of the present invention can also contain a 3' overhang. A "3' overhang" refers to at least one unpaired nucleotide extending from the 3' end of an RNA strand. Thus, in one embodiment, the siRNA of the present invention contains at least one 3' overhang that is 1 to about 6 nucleotides in length (including ribonucleotides or deoxynucleotides), preferably 1 to about 5 nucleotides in length, more preferably 1 to about 4 nucleotides in length, and particularly preferably about 1 to about 2 nucleotides in length.

[0060] When both strands of the siRNA molecule comprise 3' overhangs, the length of these overhangs can be the same or different for each strand.In the most preferred embodiment, the 3' overhangs are present on both strands of the siRNA and are 2 nucleotides in length.To enhance the stability of the siRNA of the present invention, the 3' overhangs can also be stabilized against degradation.In one embodiment, the overhangs are stabilized by including purine nucleotides such as adenosine nucleotides or guanosine nucleotides.

[0061] Alternatively, substitution of pyrimidine nucleotides with modified analogs, e.g., substitution of uridine nucleotides in the 3' overhangs with 2'-deoxythymidine, is tolerated and does not affect the efficiency of RNAi degradation. In particular, the lack of a 2' hydroxyl group in 2'-deoxythymidine significantly improves the nuclease resistance of the 3' overhangs in tissue culture medium.

[0062] The siRNAs of the present invention can target any stretch of about 18 to 30, preferably 19 to 25, contiguous nucleotides in any target mRNA sequence (including the mRNA encoding FMRP). Techniques for selecting siRNA target sequences are well known in the art. Thus, the sense strand of the siRNA of the present invention contains a nucleotide sequence identical to any contiguous stretch of about 18 to 30 nucleotides in the target mRNA.

[0063] The siRNA of the present invention can be obtained by using many techniques known to those skilled in the art.For example, siRNA can be chemically synthesized or recombinantly produced by using methods known in the art.Preferably, the siRNA of the present invention is chemically synthesized using appropriately protected ribonucleoside phosphoramidite and conventional DNA / RNA synthesizer.The siRNA can be synthesized as two separate complementary RNA molecules or as a single RNA molecule with two complementary regions. Commercially available suppliers of synthetic RNA molecules or synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colorado, USA), Pierce Chemical (part of Perbio Science, Rockford, Illinois, USA), Glen Research (Sterling, Virginia, USA), ChemGenes (Ashland, Massachusetts, USA), Qiagen (Hilden, Germany), and Cruachem (Glasgow, UK).

[0064] Alternatively, siRNA can be expressed from recombinant circular or linear DNA plasmids using any suitable promoter. Suitable promoters for expressing siRNA of the present invention from a plasmid include, for example, U6 or H1 RNA pol III promoter sequences and cytomegalovirus promoters. The selection of other suitable promoters is within the skill of those skilled in the art. The recombinant plasmids of the present invention can also contain inducible or regulatable promoters for expressing siRNA in specific tissues or specific intracellular environments. The siRNA expressed from the recombinant plasmid can be isolated from cultured cell expression systems using standard techniques or expressed intracellularly in neural cells.

[0065] The siRNA of the present invention can also be expressed in neurons from a recombinant viral vector. This recombinant viral vector contains a sequence encoding the siRNA of the present invention and any suitable promoter for expressing the siRNA sequence. Suitable promoters include, for example, the U6 or H1 RNA pol III promoter sequence and the cytomegalovirus promoter. The selection of other suitable promoters is within the skill of those in the art. The recombinant viral vector of the present invention can also contain an inducible or regulatable promoter for expressing the siRNA in the brain (e.g., hippocampal neurons), prostate, etc.

[0066] In one embodiment, the one or more siRNAs of the present invention are selected from the non-limiting group including siRNAs targeting human FMRP (S5317, S5316) from Thermo Scientific.

[0067] In one embodiment, the one or more siRNAs of the invention are selected from the non-limiting group consisting of siFMRP#1: AUAAG AGACA ACUUG GUGC (SEQ ID NO: 10) and siFMRP#2: UAACUUCGGAAUUAUGUAG (SEQ ID NO: 11).

[0068] The agent of the present invention may be selected from an antibody, an antigen-binding fragment of the antibody, or an antibody mimetic. Preferably, when the agent is an antibody, an antigen-binding fragment of the antibody, or an antibody mimetic, the agent is in the form of a plasmid or vector containing one or more nucleic acids encoding the antibody, antigen-binding fragment of the antibody, or antibody mimetic, such that the agent can be delivered into cancer cells, where FMRP is localized in the cytoplasm and nucleus.

[0069] As used herein, an "antibody" is a protein molecule that reacts with a specific antigenic determinant or epitope and belongs to one or five different classes based on structural characteristics: IgA, IgD, IgE, IgG, and IgM. Antibodies may be polyclonal (e.g., polyclonal serum) or monoclonal, including, but not limited to, fully assembled antibodies, single-chain antibodies, antibody fragments, chimeric antibodies, and humanized antibodies, provided that these molecules remain biologically active and still bind to at least one peptide of the present invention. Preferably, the antibody is a monoclonal. Also preferably, the monoclonal antibody is selected from the group including IgG1, IgG2, IgG2a, IgG2b, IgG3, and IgG4, or a combination thereof. Most preferably, the monoclonal antibody is selected from the group including IgG1, IgG2, IgG2a, and IgG2b, or a combination thereof.

[0070] A typical antibody is composed of two immunoglobulin (Ig) heavy chains and two Ig light chains. Several different types of heavy chains exist, defining the antibody's class, or isotype. These heavy chain types vary among different animals. All heavy chains contain a series of immunoglobulin domains, usually one variable (VH) domain important for antigen binding and several constant (CH) domains. Each light chain consists of two tandem immunoglobulin domains: one constant (CL) domain and one variable (VL) domain important for antigen binding.

[0071] For antibody production, various host animals may be immunized by injection with the FMRP gene product or a portion thereof (including, but not limited to, a portion of the FMRP gene product in a recombinant protein). Examples of such host animals include, but are not limited to, rabbits, mice, and rats. To enhance the immunological response, various adjuvants may be used depending on the host species. Examples include, but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

[0072] Monoclonal antibodies may be prepared using any technique which allows for the production of antibody molecules by continuous cell lines in culture, including, but not limited to, the hybridoma technique first described by Kohler and Milstein, 1975, Nature 256:495-497, the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cote et al., 1983, Proc. Natl. Acad. Sci. 80:2026-2030), and the EBV-hybridoma method (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Additionally, techniques developed for producing "chimeric antibodies" by splicing the genes for a mouse antibody molecule with the appropriate antigen specificity with the genes for a human antibody molecule with the appropriate biological activity (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-454) can be used. Alternatively, techniques described for the production of single-chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single-chain antibodies specific for one of the binding partners.

[0073] The term "isolated," when used as a modifier of an antibody of the invention, means that the antibody has been produced by the hand of man or has been completely or at least partially separated from its naturally occurring in vivo environment. Generally, an isolated antibody is substantially free of one or more materials with which it is normally associated in nature, e.g., one or more proteins. The term "isolated" does not exclude alternative physical forms of the antibody, such as multimers / oligomers, modified (e.g., phosphorylated, glycosylated, lipidated) or derivatized forms, or forms expressed in host cells produced by the hand of man.

[0074] An "isolated" antibody can also be a "substantially pure" or "purified" antibody when it is free from most or all of the materials with which it is normally associated in nature. Thus, an isolated antibody that is also substantially pure or purified will not contain a polypeptide or polynucleotide present among millions of other sequences, e.g., antibodies from an antibody library or nucleic acids from a genomic or cDNA library.

[0075] Antibody fragments that recognize specific epitopes may be generated by known techniques. "Antigen-binding fragments" include portions of full-length antibodies. Examples of antigen-binding fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, minobodies, nanobodies, linear antibodies (Zapata et al. (1995) Protein Eng. 8(10):1057-1062), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0076] Such fragments can be produced by pepsin digestion of the antibody molecule, and Fab fragments can be produced by reducing the disulfide bridges of F(ab')2 fragments. Alternatively, Fab expression libraries can be constructed (Huse et al., 1989, Science 246:1275-1281) to rapidly and easily identify monoclonal Fab fragments with the desired specificity.

[0077] Preferably, the antibodies or antigen-binding fragments thereof are engineered to penetrate cells or are directly expressed intracellularly using a gene therapy-style approach, the latter being intracellular antibodies, sometimes called intrabodies, that are produced intracellularly and bind to antigens (FMRP protein, mRNA encoding FMR, etc.) within the same cell.

[0078] The present invention also contemplates gene transfer vectors, preferably in the form of a plasmid or vector, that contain one or more nucleic acids encoding the miRNA, siRNA, piRNA, hnRNA, snRNA, sgRNA, esiRNA, shRNA, peptides or analogs thereof, antibodies or antigen-binding fragments thereof, or similar intracellular antibody mimetics, and / or antisense oligonucleotides of the present invention. As used herein, a "vector" is one that is capable of transferring a nucleic acid sequence into a target cell (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes).

[0079] Suitable vectors include SV40 and derivatives of known bacterial plasmids, such as E. coli plasmids colE1, pCR1, pBR322, pMB9 and their derivatives, RP4, and the like; phage DNA, such as many derivatives of phage X, e.g., NM989, and other phage DNA, such as M13 and filamentous single-stranded phage DNA; yeast plasmids such as the 2µ plasmid or its derivatives; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNA, such as plasmids modified to employ phage DNA or other expression control sequences; and the like.

[0080] Various viral vectors are used to deliver nucleic acids to cells in vitro or in vivo. Non-limiting examples include vectors based on herpesviruses, poxviruses, adeno-associated viruses, lentiviruses, etc. In principle, all of these are suitable for delivering expression cassettes containing expressible nucleic acid molecules encoding the miRNA, siRNA, piRNA, hnRNA, snRNA, sgRNA, esiRNA, shRNA, and antisense oligonucleotides of the present invention.

[0081] Alternatively, the gene transfer vector, preferably a viral vector, of the present invention is provided to deliver a CRISPR-based loss-of-function system comprising: i) at least one sgRNA, crRNA, and tracrRNA targeting the regulatory sequence of the FMR1 gene or the genomic DNA sequence encoding FMRP mRNA; and ii) a structure-guided endonuclease, such as an RNA-guided endonuclease. Any suitable naturally occurring or artificial RNA-guided endonuclease can be employed as long as it is effective in specifically binding the target DNA of the present invention, and may be selected from the non-limiting group including Cas9, Cpf1, and FEN-1. Preferably, the RNA-guided endonuclease is Cas9.

[0082] In a preferred embodiment, the viral vector is an adenoviral vector, preferably a lentiviral vector or a baculoviral vector, most preferably an adenoviral / adeno-associated viral (AAV) vector, although other delivery means or vehicles are known (e.g., yeast systems, microvesicles, gene guns / vectors attached to gold nanoparticles, etc.), and in some embodiments, one or more of the viral or plasmid vectors may be delivered via liposomes, nanoparticles, exosomes, microvesicles, or gene guns. More preferably, the viral vector is selected from the group including adeno-associated viruses (AAV) and lentiviruses. Lentiviruses can be first-, second-, or third-generation.

[0083] The present invention also contemplates a host cell comprising one or more nucleic acids encoding the plasmid or vector of the present invention, or the miRNA, siRNA, piRNA, hnRNA, snRNA, sgRNA, esiRNA, shRNA, CRISPR-based loss-of-function system, and / or antisense oligonucleotide of the present invention. The host cell can be any prokaryotic or eukaryotic cell, preferably the host cell is a eukaryotic cell, and most preferably the host cell is a mammalian cell. The host cell of the present invention can deliver the plasmid or vector of the present invention to cancer cell(s) using many techniques known to those skilled in the art, such as exosomes and microvesicles.

[0084] Provided herein is a pharmaceutical composition comprising: i) a therapeutically effective amount of an agent that modulates the expression and / or activity of an FMRP protein, mRNA encoding FMRP, and / or FMR1 gene as described herein, or ii) a plasmid or vector of the invention, or iii) a host cell of the invention; a pharmaceutically acceptable carrier or diluent; Also provided is a pharmaceutical composition comprising:

[0085] The pharmaceutical compositions of the present invention may be prepared in a manner known per se, for example, by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Suitable formulations depend on the chosen route of administration. Techniques for formulating and administering the compounds of the present application may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pennsylvania, latest edition.

[0086] Any agent of the present invention, either by itself or in a pharmaceutical composition mixed with a suitable carrier or excipient(s), can be administered to animals, including human patients, in therapeutically effective doses to treat or ameliorate a variety of disorders, including disorders characterized by insufficient, abnormal, or excessive FMRP activity.

[0087] In some embodiments, the pharmaceutical compositions of the present invention are useful for treating and / or preventing cancer and / or cancer metastasis in a subject in need thereof.

[0088] As used herein, the term "therapeutically effective amount" refers to an amount of an agent that modulates the expression and / or activity of FMRP protein, mRNA encoding FMRP, and / or FMR1 gene, within the scope of sound medical judgment, high enough to significantly favorably alter the symptoms and / or condition to be treated, yet low enough to avoid serious side effects (a reasonable risk / benefit ratio).

[0089] A therapeutically effective amount of an agent that modulates the expression and / or activity of FMRP protein, mRNA encoding FMRP, and / or the FMR1 gene is selected depending on various factors, such as the type, species, age, weight, sex, and condition of the patient, the severity of the condition being treated, the route of administration, the patient's renal and hepatic function, etc. One skilled in the art can readily determine and prescribe the effective amount of the agent needed to prevent, combat, or inhibit the progression of cancer and / or cancer metastasis.

[0090] "Pharmaceutically acceptable carrier or diluent" means a carrier or diluent that is generally safe, non-toxic, and useful for preparing a desired pharmaceutical composition, and includes a carrier or diluent that is acceptable for human pharmaceutical use.

[0091] Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

[0092] Pharmaceutically acceptable excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like.

[0093] The pharmaceutical composition may further contain one or more pharmaceutically acceptable salts, such as mineral acid salts, e.g., hydrochloride, hydrobromide, phosphate, sulfate, etc.; or organic acid salts, e.g., acetate, propionate, malonate, benzoate, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffers, gels or gelling materials, flavors, colorants, microspheres, polymers, suspending agents, etc., may also be present in the pharmaceutical composition. Additionally, particularly when the dosage form is reconstitutable, one or more other conventional pharmaceutical ingredients, such as preservatives, wetting agents, suspending agents, surfactants, antioxidants, anti-caking agents, fillers, chelating agents, coating agents, chemical stabilizers, etc., may also be present. Suitable exemplary ingredients include macrocrystalline cellulose, sodium carboxymethylcellulose, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, gelatin, albumin, and combinations thereof. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ, 1991), which is incorporated herein by reference.

[0094] In some embodiments, the present invention relates to the identification, production, and use of agents that modulate FMRP gene expression or the activity of FMRP gene products, including, but not limited to, nucleic acids encoding FMRP and homologs, analogs, and deletions thereof, as well as chemical compounds, peptides or analogs thereof, antibodies or antigen-binding fragments thereof, antibody mimetics, or nucleic acids, and pharmaceutical formulations and routes of administration of such compounds.

[0095] Assays using FMRP transfectants can be successfully used to identify agents that modulate FMRP gene expression. Assays for the activity of the FMRP gene product are also described.

[0096] The invention also includes antisense oligonucleotides specific for FMRP transcripts, antibodies (fragments and mimetics) against the gene product, cell lines engineered to stably express FMRP, assays for screening compounds, including peptides, polynucleotides, and small organic molecules, to identify those that inhibit the expression or activity of the FMRP gene product, and methods of using such compounds to treat diseases characterized by FMRP activity.

[0097] Human FMRP protein will be useful for in vitro studies of the mechanism of action of human FMRP, particularly for further studies of the mechanism of action of FMRP-selective inhibitors identified through drug screening, or for studies of the mechanism of action of existing drugs or inhibitors that may be identified by other means. Purified human FMRP protein will also be useful for producing crystals suitable for X-ray crystallography. Such crystals will be extremely valuable for the rational design of drugs based on molecular structure.

[0098] The present invention provides an in vitro system for screening drugs that modulate FMRP stability and / or activity. Assays can be performed on live mammalian cells, which more closely resemble the effects of specific serum levels of drugs in vivo, or on microsomal extracts prepared from cultured cell lines. Studies using microsomal extracts may allow for more rigorous determination of direct interactions.

[0099] Thus, the present invention also provides a method for assessing the relative inhibitory activity of an agent that selectively inhibits FMRP, comprising, for example, contacting a transgenic cell line expressing FMRP or a microsomal extract thereof with a preselected amount of the agent in an appropriate culture medium or buffer, adding arachidonic acid to the mixture, and measuring the level of FMRP synthesis or FMRP protein activity by the cell line or microsomal extract relative to a portion of a control cell line or microsomal extract in the absence of the agent.

[0100] In some embodiments, the invention provides a method for determining the ability of an agent to inhibit FMRP activity in a cell, comprising: (1) adding a first preselected amount of the agent to cells in culture medium, the cells containing a DNA sequence that expresses FMRP; (2) measuring the level of FMRP activity by the cells; (3) comparing the level with the level of FMRP activity by the cell line in the absence of the agent; The present invention provides a method comprising:

[0101] In some embodiments, the cell is a transgenic cell. In some embodiments, the cell is a transgenic cell line. In some embodiments, the transgenic cell or transgenic cell line comprises a cell that contains a chromosomally integrated recombinant DNA sequence that expresses FMRP. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell does not express its own FMRP activity.

[0102] In some embodiments, the cells are human or mouse cancer cells that significantly upregulate (increase) FMRP expression during tumor formation and malignant progression, thereby conferring resistance to immune attack.

[0103] In some embodiments, the FMRP is a mammalian FMRP, preferably a human FMRP.

[0104] In some embodiments, the level of expression and / or activity of FMRP is determined by an agent that inhibits the binding of FMRP to its target RNA.

[0105] In some embodiments, the present invention provides a method for identifying an agent that modulates the expression and / or activity of i) FMRP protein, ii) mRNA encoding FMRP, and / or iii) the FMR1 gene, comprising: (1) providing a sample expressing FMRP; (2) contacting the biological sample with a test agent; (3) determining the level of expression and / or activity of FMRP; (4) comparing the level of expression and / or activity with a control sample that has not been contacted with the test agent; (5) selecting a test agent that reduces the level of expression and / or activity of the FMRP; The present invention provides a method comprising:

[0106] In some embodiments, the sample is a cell that naturally expresses high levels of endogenous FMRP. In some embodiments, the sample is a cell that has been engineered to express FMRP.

[0107] In some embodiments, the cell is a transgenic cell. In some embodiments, the cell is a transgenic cell line. In some embodiments, the transgenic cell or transgenic cell line comprises a cell that contains a chromosomally integrated recombinant DNA sequence that expresses FMRP. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell does not express its own FMRP activity.

[0108] In some embodiments, the cells are human or mouse cancer cells that significantly upregulate FMRP expression during tumorigenesis and malignant progression, conferring resistance to immune attack.

[0109] In some embodiments, the FMRP is a mammalian FMRP, preferably a human FMRP.

[0110] In some embodiments, the level of expression and / or activity of FMRP is determined by an agent that inhibits the binding of FMRP to its target RNA.

[0111] Agents identified in the screens exhibit the ability to selectively modulate the expression and / or activity of FMRP, including, but not limited to, nucleic acids encoding FMRP and homologs, analogs, and deletions thereof, as well as chemical compounds, peptides or analogs thereof, antibodies or antigen-binding fragments thereof, antibody mimetics, or nucleic acids.

[0112] The DNA of the present invention encoding the FMRP gene, or a homologue, analogue or fragment thereof, may be used in accordance with the present invention to diagnose a condition that is a phenotype of FMRP genotype or expression of FMRP.

[0113] Alternatively, the pharmaceutical composition of the present invention further comprises one of multiple components of an anti-cancer therapy. Preferably, the anti-cancer therapy comprises a therapeutically effective amount of an immune checkpoint inhibitor. Preferably, the immune checkpoint inhibitor is selected from the group including a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor, or a combination thereof. Alternatively, or additionally, the one or more anti-cancer therapies is a chemotherapeutic agent or a cocktail of multiple different chemotherapeutic agents, as described herein.

[0114] As used herein, "PD-1 inhibitor" refers to any agent that interferes with or blocks the binding of the PD-1 receptor on T cells to its ligands, PD-L1 and PD-L2, present on tumor cells. A PD-1 inhibitor may be an antibody or fragment thereof that interferes with, inhibits, or blocks the binding of PD-1 to its ligands. A PD-1 inhibitor may be a small molecule or other agent. Non-limiting examples of PD-1 inhibitors include nivolumab, pembrolizumab, pidilizumab, BMS 936559, MPDL3280A, MSB0010718C BGB-108, and mDX-400 and MEDI4736.

[0115] Non-limiting examples of PD-L1 inhibitors are selected from the group including MEDI-0680, RG-7446, durvalumab, KY-1003, KD-033, MSB-0010718C, TSR-042, ALN-PDL, STI-A1014 and BMS-936559.

[0116] As used herein, "CTLA-4 inhibitor" means any agent that interferes with or blocks CTLA-4, such as anti-CTLA-4 mAbs or blockers, for example, ipilimumab, tremelimumab, and abatacept.

[0117] In another embodiment, the FMRP-directed anti-cancer therapy is included in combination with a therapeutically effective amount of an anti-tumor vaccine comprising a personalized neoantigen cocktail or other immunostimulatory agent that enhances the anti-tumor immune response.

[0118] The present invention further provides a method for treating and / or preventing cancer and / or cancer metastasis in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the present invention, alone or in combination with one or more anti-cancer therapies. Most preferably, the anti-cancer therapy comprises a therapeutically effective amount of an immune checkpoint inhibitor. Preferably, the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor, or a combination thereof. Alternatively, or additionally, the anti-cancer therapy is a chemotherapeutic agent described herein, or a cocktail of multiple different chemotherapeutic agents.

[0119] It will be understood that the combination of the pharmaceutical composition of the present invention and the PD-1 / PD-L1 / CTLA-4 inhibitor may be administered in any order or simultaneously. In selected embodiments, the pharmaceutical composition and PD-1 / PD-L1 / CTLA-4 are administered to a patient who has previously been treated with other anti-cancer agents. In certain other embodiments, the pharmaceutical composition and PD-1 / PD-L1 / CTLA-4 inhibitor are administered substantially simultaneously or concomitantly. For example, a subject may be given a pharmaceutical composition of the present invention while undergoing a course of treatment with a PD-1 / PD-L1 / CTLA-4 inhibitor. In addition, it is contemplated that the subject may already be undergoing or may be concurrently undergoing other forms of cancer treatment, such as chemotherapy. In certain embodiments, the pharmaceutical composition of the present invention is administered within one year of treatment with the PD-1 / PD-L1 / CTLA-4 inhibitor. In certain alternative embodiments, the pharmaceutical compositions of the present invention are administered within 10 months, 8 months, 6 months, 4 months, or 2 months of any treatment with the PD-1 / PD-L1 / CTLA-4 inhibitor and / or additional anti-cancer therapy. In certain other embodiments, the pharmaceutical compositions of the present invention are administered within 4 weeks, 3 weeks, 2 weeks, or 1 week of any treatment with the PD-1 / PD-L1 / CTLA-4 inhibitor and / or additional anti-cancer agent or therapy. In some embodiments, the pharmaceutical compositions of the present invention are administered within 5 days, 4 days, 3 days, 2 days, or 1 day of any treatment with the PD-1 / PD-L1 / CTLA-4 inhibitor and / or additional anti-cancer therapy or agent. It will further be appreciated that the pharmaceutical compositions of the present invention and the PD-1 / PD-L1 / CTLA-4 inhibitor and / or additional anti-cancer agent or therapy may be administered to a subject within hours or minutes (i.e., substantially simultaneously).

[0120] In embodiments, the agents of the invention could be combined with other immunomodulatory agents that sustain the killing activity and abundance of T cells (and NK cells) or disrupt other barriers such as myeloid-derived suppressor cells (MDSCs) and immunosuppressive macrophages, insofar as they complement the effects of inhibiting FMRP.

[0121] In some embodiments, the agents of the invention could be combined with an ADAR1 inhibitor: knockout (i.e., genetic inhibition) of ADAR1, an immunosuppressive RNA-editing enzyme, has a combinatorial benefit in extending overall survival in double-KO tumors that also have a knockout of FMRP.

[0122] Anti-cancer agents that may be administered in combination with the pharmaceutical compositions of the present invention and PD-1 / PD-L1 / CTLA-4 inhibition include chemotherapeutic agents. Thus, in some embodiments, the methods or treatments involve the combined administration of the pharmaceutical compositions of the present invention and PD-1 / PD-L1 / CTLA-4 inhibitors with a chemotherapeutic agent or a cocktail of multiple different chemotherapeutic agents. Treatment with the pharmaceutical compositions of the present invention can precede, be concurrent with, or follow the administration of these other therapies. Chemotherapeutic agents contemplated by the present invention include chemical entities or drugs known in the art and commercially available, such as gemcitabine, irinotecan, doxorubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxol, methotrexate, cisplatin, melphalan, vinblastine, and carboplatin. Combined administration (concomitant administration) can include simultaneous administration in a single pharmaceutical formulation or in separate formulations, or sequential administration, which may be in any order, but generally within a time period that allows all active agents to simultaneously exert their biological activity. Preparation and administration schedules for such chemotherapeutic agents can be used according to the manufacturer's instructions or as determined empirically by one of skill in the art.

[0123] Chemotherapeutic agents useful in the present invention include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkylsulfonates such as busulfan, impresulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylmelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; chlorambucil, chlornaphtha, and the like. Nitrogen mustards such as benzodiazepine, colofosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novoenbichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; aclacinomycins, actinomycin, and ausrama Isin (anthramycin, authramycin), azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, ezorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, Antibiotics such as potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine;Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenal hormones such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid replenisher; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fename acetaminophen; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; schizofuran (schizofiran); spirogermanium; tenuazonic acid; triaziconazole; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL, Bristol-Myers Squibb); Squibb Oncology (Bristol-Myers Squibb Oncology, Princeton, New Jersey) and doxetaxel (TAXOTERE, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine;Also included are, but are not limited to, navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronic acid; CPT11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (OMFO); retinoic acid; esperamicin; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Chemotherapeutic agents also include antihormonal agents that regulate or inhibit hormone action on tumors, such as antiestrogens such as tamoxifen, raloxifene, aromatase inhibitors such as 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston), and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0124] In certain embodiments, the therapeutic agent is a kinase inhibitor.In certain embodiments, this kinase inhibitor is a multi-target receptor tyrosine kinase inhibitor.Kinase inhibitors include but are not limited to sunitinib, pazopanib, crizotinib, and dasatinib.In certain embodiments, the second anticancer agent is sunitinib.

[0125] In certain embodiments, the therapeutic agent is an inhibitor of mammalian target of rapamycin (mTOR). mTOR inhibitors include but are not limited to temsirolimus, sirolimus, deforolimus, and everolimus. In certain embodiments, the second anticancer agent is everolimus.

[0126] In certain embodiments, the therapeutic agent is a somatostatin analog. Somatostatin analogs act through interaction with specific, high-affinity membrane receptors for somatostatin. Somatostatin analogs include, but are not limited to, octreotide, somatuline, and RC160 (octastatin). In certain embodiments, the second anticancer agent is octreotide.

[0127] In certain embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. A topoisomerase inhibitor is a chemotherapeutic agent that interferes with the action of a topoisomerase enzyme (e.g., topoisomerase I or II). Examples of topoisomerase inhibitors include, but are not limited to, doxorubicin hydrochloride, daunorubicin citrate, mitoxantrone hydrochloride, actinomycin 0, etoposide, topotecan hydrochloride, teniposide (VM-26), and irinotecan. In certain embodiments, the second anticancer agent is irinotecan.

[0128] In certain embodiments, the chemotherapeutic agent is an alkylating agent. In certain embodiments, the chemotherapeutic agent is temozolomide.

[0129] In certain embodiments, the chemotherapeutic agent is an antimetabolite. An antimetabolite is a chemical compound that has a structure similar to a metabolite necessary for normal biochemical reactions, but different enough to interfere with one or more normal cellular functions, such as cell division. Antimetabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine (GlaxoSmithKline), 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these. In certain embodiments, the second anticancer agent is gemcitabine. In certain embodiments, the tumor to be treated is a pancreatic neuroendocrine tumor and the second anti-cancer agent is an antimetabolite (eg, gemcitabine).

[0130] In certain embodiments, the chemotherapeutic agent is an antimitotic agent, including, but not limited to, agents that bind tubulin. As a non-limiting example, the agent includes a taxane. In certain embodiments, the agent includes paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In certain embodiments, the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel (e.g., ABRAXANE), DHA-paclitaxel, or PG-paclitaxel. In certain alternative embodiments, the antimitotic agent includes a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, or vindesine, or a pharmaceutically acceptable salt, acid, or derivative thereof. In some embodiments, the antimitotic agent is an inhibitor of Eg5 kinesin or an inhibitor of a mitotic kinase such as Aurora A or Plk1.

[0131] In certain embodiments, the treatment involves the combined administration of a pharmaceutical composition of the invention, a PD-1 / PD-L1 / CTLA-4 inhibitor described herein, and radiation therapy. Treatment with the pharmaceutical composition of the invention can occur prior to, simultaneously with, or after the administration of radiation therapy. Any administration schedule for such radiation therapy can be used as determined by one of skill in the art.

[0132] In another aspect of the invention, the pharmaceutical compositions of the invention are sustained release formulations or formulations administered using sustained release devices. Such devices are well known in the art and include, for example, transdermal patches and miniature implantable pumps that can provide continuous, steady-state drug delivery over time at various doses to achieve a sustained release effect in non-sustained release pharmaceutical compositions.

[0133] In another aspect of the invention, the pharmaceutical composition of the invention is administered to said patient before, during and / or after radiation therapy.

[0134] "Radiation therapy" refers to the use of high-energy radiation to shrink tumors and kill cancer cells. Examples of radiation therapy include, but are not limited to, external radiation therapy and internal radiation therapy (also called brachytherapy).

[0135] External radiation therapy is the most common and typically involves directing a beam of ionizing radiation, either direct or indirect, at the tumor or cancer site. The radiation beam, photon, cobalt, or particle beam treatment, is focused on the tumor or cancer site, but exposure of normal, healthy tissue is nearly impossible to avoid. The energy source for external radiation therapy is selected from the group including direct or indirect ionizing radiation (e.g., X-rays, gamma rays, particle beams, or a combination thereof).

[0136] Internal radiation therapy involves implanting radiation-emitting sources, such as beads, wires, pellets, or capsules, into the body at or near the tumor site. The energy source for internal radiation therapy is selected from the group of radioisotopes, including iodine (iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, indium, phosphate, or cobalt radioisotopes, and combinations thereof. Such implants can be removed after treatment or left in place. Types of internal radiation therapy include, but are not limited to, interstitial and intracavitary brachytherapy (high-dose rate, low-dose rate, and pulsed-dose rate).

[0137] Currently, less common forms of internal radiation therapy involve biological carriers of radioisotopes, such as radioimmunotherapy, in which patients are given tumor-specific antibodies conjugated to radioactive material. These antibodies bind to tumor antigens, thereby effectively delivering the radiation dose to the relevant tissue.

[0138] Methods for administering radiation therapy are well known to those skilled in the art.

[0139] The pharmaceutical compositions of the present invention may be administered to a subject by different routes, including, but not limited to, orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, by inhalation, buccal administration, intrathoracically, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intratumorally, intrathecally, and intraarticularly, or combinations thereof. For human use, the compositions may be administered in an appropriately acceptable formulation according to normal human usage. One skilled in the art will readily determine the most appropriate method and route of administration for a particular patient. The compositions of the present invention may also be administered by conventional syringes, needleless injection devices, "microprojectile bombardment guns," or other physical methods such as electroporation ("EP"), "hydrodynamic methods," or ultrasound.

[0140] The pharmaceutical compositions of the present invention may also be delivered to patients by several techniques, including DNA injection (also called DNA vaccination), with or without in vivo electroporation, liposome-mediated, nanoparticle-facilitated recombinant vectors, such as the recombinant lentiviruses, recombinant adenoviruses, and recombinant adenovirus-associated viruses described herein. The compositions may be injected intravenously, locally injected into the brain or muscle, or electroporated into tissues of interest, such as muscle, brain, liver, prostate, breast, kidney(s), hematopoietic system, etc.

[0141] The agents and / or pharmaceutical compositions of the present invention may be used in any method in which it is beneficial to modulate the expression and / or immunosuppressive activity of i) FMRP protein, ii) mRNA encoding FMRP protein, and / or iii) the FMR1 gene encoding FMRP.

[0142] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications within the spirit or essential characteristics thereof. The invention also includes all steps, features, compositions, and compounds referred to or illustrated herein, individually or collectively, and any and all combinations of any two or more of such steps or features. Therefore, the present disclosure is considered to be non-limiting in all exemplified aspects, and the scope of the invention is indicated by the appended claims, with all modifications that fall within the spirit and range of equivalents intended to be embraced therein. Various documents are cited throughout this specification, each of which is incorporated herein by reference in its entirety. The foregoing description will be more fully understood with reference to the following examples. [Example]

[0143] Example 1 Materials and Methods Generation of Crisper-edited tumor cell lines Mouse PDAC 4361.12 cells were cultured in DMEM containing 10% FBS. The FMR1 gene was knocked out in cells using the CRISPR / Cas9 system. Cells were transiently transfected with two Cas9 and single guide RNA (sgRNA) expression plasmids and selected with blasticidin for 5 days. This transient CRISPR strategy for knocking out FMRP prevents potential nonspecific effects mediated by stable integration of Cas9 / sgRNA into the genome. The guide sequence used for FMR1 was 5'-GTGGAAGTGCGGGGCTCCAA-3' (SEQ ID NO: 12) or 5'-GAGCTGGTGGTGGAAGTGCG-3' (SEQ ID NO: 13). Cells were plated single-cell in 96-well plates without blasticidin. Knockout clones were selected for FMRP protein loss by immunoblotting. The resulting FMRP KO cells retained sensitivity to blasticidin, indicating that the CRISPR / Cas9 system was only transiently expressed in these cells. Two independent KO clones and a WT clone (transfected with the Cas9 vector and empty SgRNA vector) were analyzed as indicated.

[0144] Animal experiments All animal experiments were performed in accordance with protocols approved by the local animal experimentation committee of Canton de Vaud (license number 3214). FVBn, Balb / c, C57B / 6, NSG, or SCID / beige mice were used at 8 weeks of age. For lung metastasis assays, 2 × 10 cells suspended in 200 μl PBS were used. 5 Mouse PDAC WT or FMRP KO cells were injected into the lateral tail vein of mice. For primary tumor growth assays, 5 x 10 cells suspended in 100 μl of PBS were injected. 5 The cells were injected subcutaneously into mice.

[0145] Analysis of tumor-infiltrating lymphocytes by flow cytometry Flow cytometry was performed using a BD LSRII Fortessa, and results were analyzed using FlowJo software (Treestar). Primary tumor cell suspensions were blocked with mouse Fc block (anti-CD16 / CD32; Biolegend, #101312) before staining. Fluorescently conjugated anti-mouse CD45 (clone 30F-11), CD3e (clone 145-2C11), CD8a (clone 53-6.7), granzyme B (clone NGZB), TNFα (MP6-XT22), and IFNγ (clone XMG1.2) antibodies were used according to the manufacturer's protocol. Blue UV was used to stain dead cells. To analyze intracellular cytokine expression, mice bearing sc tumors were injected IP with 250 μg of the protein transport inhibitor brefeldin A (BD Biosciences, #555029) 6 h before sacrifice. Then, intracellular cytokine staining was performed using a Fixation / Permeabilization Solution Kit (Sigma, B6542-25MG).

[0146] Immunohistochemical and immunofluorescent staining The collected mouse tissues were fixed overnight in 4% paraformaldehyde, embedded in paraffin, and then sectioned using a microtome (Leica). Antigen retrieval was performed using citrate buffer (pH = 6.0) for 20 minutes in a 95°C water bath or Tris-EDTA buffer (pH = 8.0) for 10 minutes in a 95°C water bath. Primary antibodies were incubated overnight at 4°C. For immunohistochemistry (IHC) staining, secondary antibodies (ImmPRESS HRP Reagent Kit, anti-rabbit MP-7401 and anti-rat MP-7444) were incubated for 45 minutes at room temperature, and finally visualized using the peroxidase substrate DAB (Sigma-Aldrich, D5637-1G) for the same time (maximum 10 minutes) at room temperature. Stained tissue sections were counterstained with Mayer's hematoxylin. For immunofluorescence (IF) staining, secondary antibodies (Alexa Fluor 488, 568, and 647, Thermo Fisher Scientific) were incubated for 45 minutes at room temperature. Images were acquired using a Leica DM5500B and a Zeiss LSM 700 upright confocal microscope and analyzed with Image J. The antibodies used were as follows: FMRP, Abcam, ab191411; mouse CD8, Thermo Fisher Scientific, 14-0808-82.

[0147] statistical analysis Statistical analysis was performed using Prism 7 (GraphPad Software). Unless otherwise stated, Student's t-test (two-tailed) was used for non-paired experiments. Wilcoxon signed-rank test (two-tailed) was used for paired experiments that did not follow a Gaussian distribution. P < 0.05 was considered statistically significant. Values are means ± SEM.

[0148] result Recent studies have revealed that FMRP acts as a downstream effector of NMDAR signaling and promotes the invasive development of pancreatic cancer. 16We further confirmed that FMRP expression is elevated in human (data not shown) and mouse PDAC tissues (Fig. 1A).

[0149] To further investigate the role of FMRP in tumor progression, we used the Crisper / Cas9 system. 10 P48-cre;LSL-Kras in an FvBn background was used. G12D ;P53 R172H / + We knocked out FMRP in the murine pancreatic adenocarcinoma (PDAC) cell line 4361.12, a single-cell derived cell line from a PDAC mouse model (Figure 1B). Importantly, we performed a transient Crisper strategy to knock out FMRP to avoid potential nonspecific effects and immunogenicity that may arise from stable integration of Cas9 / sgRNA into the genome.

[0150] In vitro functional assays showed no significant difference in colony-forming ability between WT and FMRP KO PDAC cells (Figure 1C). Considering the possible role of FMRP in metastasis, we first performed a standard in vivo lung metastasis assay by injecting WT and FMRP KO cells into the tail vein of immunocompetent FVBn mice (Figure 1D). Surprisingly, two of the five mice injected with FMRP-KO2 cells survived for 120 days after injection, whereas all five mice injected with FMRP-WT cells died before 25 days after injection (Figure 1E). When we performed a similar in vivo lung metastasis assay in immunodeficient SCID / beige mice, we found no significant difference in overall survival between these two groups (Figure 1F), suggesting that the adaptive immune system is responsible for the improved survival observed in mice bearing FMRP-KO tumors. Next, we used a primary tumor model formed by subcutaneous injection of cancer cells (Figure 1H). When FMRP-KO cells were subcutaneously injected into FVBn mice, tumor growth of FMRP-KO cells was significantly impaired compared with that of FMRP-WT cells. However, when the cells were subcutaneously injected into immunodeficient NSG mice, no significant difference in tumor weight was observed between the two groups (Figures 1I and 1J), further suggesting that FMRP may be involved in regulating antitumor immunity in vivo.

[0151] Importantly, IHC staining of tumors formed from WT and FMRP-KO cancer cells revealed that KO tumors were infiltrated with numerous CD8+ cytotoxic T cells. In stark contrast, CD8+ T cells were almost absent in WT tumors (Figure 2A). The number of CD45+ immune cells was also increased in FMRP-KO tumors compared with WT tumors (Figure 2B). Consistent with this, FACS analysis of primary cell suspensions from WT and FMRP-KO tumors further demonstrated a dramatic increase in the numbers of CD45+ immune cells, CD3+CD8+ T cells, and GRZb+, IFNγ+, and TNF+ T cells in KO tumors compared with WT tumors (Figure 2C–G), further supporting the role of FMRP in suppressing antitumor immunity in vivo. Furthermore, dual IHC staining of FMRP and CD8 in mouse PDAC tissues revealed that CD8 T cells were barely detectable in the center of FMRP-expressing tumors (Figure 2H). A significant inverse correlation between FMRP expression and CD8 T cell infiltration was also found in human PDAC samples.

[0152] We investigated whether deletion of FMRP in cancer cells alters the expression of immune checkpoint proteins and induces antitumor immune responses. If FMRP-suppressed antitumor immunity depends on established T cell co-inhibitory PD-1 or possibly CTLA-4 signaling, we expected downregulation of PD-1 ligands PD-L1 / CD274 and PD-L2 / PDCD1LG2, or CTLA-4 ligands B7 / B7-1 / CD80 and CD86, in FMRP-KO tumor cells. However, WB analysis and IHC staining showed that PD-L1 expression was unchanged in WT and FMRP-KO cells in vitro and in vivo (Figures 3A-B), and PDL2, CD86, and CD80 were barely detectable (data not shown). This suggests that the underlying mechanism of FMRP-mediated immune resistance in this PDAC cancer cell line does not involve suppression of immune checkpoint ligands PD-1 or CTLA-4.

[0153] Additionally, we performed preclinical studies using anti-PD1 antibodies against WT tumors formed in immunocompetent FVBn mice. These tumors were found to be unresponsive to anti-PD1 therapy, recapitulating the unresponsiveness of human PDAC patients to anti-PD1 therapy (Figure 3C). Despite this resistance to anti-PD1 checkpoint immunotherapy, FMRP-KO derivative PDAC tumor cells exhibited significantly impaired tumor growth in vivo (Figure 1), which was associated with the influx of CD8 T cells (Figure 2). These results suggest that FMRP inhibitors could be used as a novel immunotherapeutic strategy for the treatment of PDAC and other tumors refractory to checkpoint inhibitors. Interestingly, PD1 antibody treatment of FMRP-KO tumors further suppressed tumor growth, indicating that combining anti-PD1 antibodies with FMRP knockout may offer a combinatorial benefit in extending survival (Figure 3D).

[0154] Recent studies have shown that ADAR1, an RNA-binding protein that mediates A-to-I RNA super-editing, promotes resistance to immune checkpoint blockade. 23Interestingly, another study showed that FMRP also regulates RNA hyperediting in neuronal cells by physically interacting with ADAR1. The strong interaction between FMRP and ADAR1 in mouse PDAC cells was confirmed by co-immunoprecipitation (co-IP) and reverse co-IP (Figure 4A-B). To investigate whether the combination of FMRP and ADAR1 double knockout induces even stronger antitumor immune responses, ADAR1 single knockout and FMRP / ADAR1 double knockout PDAC cells were generated using transient transfection of Cas9 / sgRNA vectors targeting the FMR1 and ADAR1 genes (Figure 4C) and injected into syngenic mice. Interestingly, combined knockout of FMRP and ADAR1 significantly extended overall survival compared with the FMRP-only knockout and ADAR1-only knockout groups (Figure 4D). This supports the clinical application of combined targeting of FMRP and ADAR1 in cancer immunotherapy.

[0155] To broadly explore the potential role of FMRP in suppressing antitumor immunity in other cancer types, we detected dramatically increased FMRP expression in mouse colon tissues (Figure 5A), melanoma tissues (Figure 6A), pancreatic neuroendocrine tumors (PNETs) and liver metastases (Figure 7A), and breast cancer tissues (Figure 8A) compared with corresponding normal tissues. Human data are consistent with mouse data (not shown). Similarly, FMRP KO in mouse colon cancer cells (Figure 6B) and melanoma cells (Figure 7B) did not significantly impair in vitro colony formation (reflecting proliferation and survival capacity). However, FMRP KO significantly impaired colon tumor growth in syngenic, but not immunodeficient, mice (Figures 6D-6I, 7D-7E). Increased CD8 T cell numbers were observed only in FMRP KO colon tumors, but not in WT tumors (Figure 6G). FMRP was also knocked out in PanNET tumors arising in the RIP1-Tag2 (RT2) PanNET mouse model by crossing RIP-7 cre mice with RT2 mice and FMR1-floxed mice. Importantly, FMRP KO RT2 mice had significantly longer survival times than WT RT2 mice, strongly supporting the involvement of FMRP in promoting PNET tumor progression.

[0156] Additional investigations of FMRP expression in human tumors revealed significant upregulation in 30–100% of patients with a wide range of cancer types, including all major forms of lethal solid tumors (see, e.g., Figure 11 ).

[0157] Example 2 Directly targeting the RNA-binding site of FMRP using oligonucleotides and peptides to inhibit interactions involved in FMRP effector functions The RGG and KH2 RNA-binding domains of FMRP have been implicated in the functional activity of FMRP in several studies (e.g., Vasilyev, 2015; Darnell, 2005). Based on structural knowledge of these interactions, it is possible to inhibit the FMRP-RNA interaction by delivering abundant competitor molecules. In one variation of this method, DNA or RNA oligonucleotides representing core sequences from sc1 / kc RNA that bind to the RGG / KH2 domains, respectively, are synthesized. In some embodiments, locked nucleic acid (LNA) technology can be included in the synthesis to increase both the half-life of the oligonucleotide and its binding affinity. In a second variation, a polypeptide spanning the RGG and KH2 domains of FMRP is synthesized and tested. In some embodiments, the polypeptides are linked by a polypeptide linker. In either variation, candidates would first be tested by delivery to cultured cancer cells expressing FMRP and scored for impaired invasiveness in a Boyden chamber assay as previously described (Li and Hanahan, 2013; Li, Zeng et al., 2018) and shown in Figure 9. Candidate compounds would be inoculated into tumors composed of cancer cells expressing FMRP as described herein, and the resulting infiltration of CD8 T cells, which would otherwise be excluded by FMRP expression, would be assessed. A variation of this method would involve the use of transfection enhancers to increase tumor uptake of candidate oligonucleotides.

[0158] Example 3 Therapeutic suppression of FMRP by siRNA An increasingly well-validated therapeutic strategy involves delivering siRNAs that bind to and destabilize or block mRNA translation to tissues to suppress the production of disease-related proteins (Selvam, 2017). In this approach, siRNAs are designed to bind to and inhibit FMR1 mRNA (encoding FMRP) and are assayed by delivery to FMRP tumors lacking CD8 T cells. The invasion of such cells is scored using gene knockout tumors described elsewhere in this application as a benchmark. Prior to such in vivo testing, candidate siRNAs (i.e., siCtrl: UAAGG CUAUG AAGAG AUAC (SEQ ID NO: 9); siFMRP#1: AUAAG AGACA ACUUG GUGC (SEQ ID NO: 10); and siFMRP#2: UAACUUCGGAAUUAUGUAG (SEQ ID NO: 11)) were tested in a cancer cell invasion assay, where the inhibitory capacity of prototype siRNAs against FMR1 mRNA is depicted in Figure 9. In some embodiments, siRNAs against FMR1 could include additional refinements, for example, by chemical modifications to enhance stability and activity (Hassler, 2018) and / or the use of "transfection" reagents to enhance delivery of the nucleic acid to cancer cells within the tumor.

[0159] Example 4 Description of a high-throughput biochemical screening method to identify small molecules that bind to key interaction sites on FMRP One well-described mode of FMRP interaction involves the regulation of a select set of mRNAs containing a G4 structural motif that binds to a domain called RGG on the FMRP protein. Binding of this G4 structural motif alters the translation of the target mRNA. The RGD domain of FMRP tightly binds an RNA called sc1, which is widely used as a prototype for FMRP binding to target mRNAs. Therefore, compounds that inhibit the binding of sc1 RNA to FMRP are considered to represent a) inhibitors of the translational control mechanism involving FMRP binding to mRNA, and b) tight binders of the RGG site that do not necessarily inhibit all functions of FMRP, in case other mechanisms of action of FMRP other than the RGG domain are involved in the newly discovered immunosuppressive activity of FMRP.

[0160] A second mode of FMRP interaction involves binding to high-affinity RNA targets via the KH2 RNA-binding domain (Darnell et al., 2005). Previous studies have identified a series of RNAs with structural and sequence-specific features, termed "kissing complex (kc) RNAs." For example, kc2 RNA can displace FMRP from polyribosomes at half-maximal concentrations of approximately 100 nM. Human and mouse studies have shown that a single missense mutation in KH2 abrogates FMRP binding to polysomes and causes a severe form of fragile X syndrome. Compounds that inhibit kcRNA binding to FMRP could disrupt polysome association and abolish FMRP function. Such compounds could be drugs, such as those disclosed herein. In some embodiments, the drug is a synthetic nucleic acid that inhibits kcRNA binding to FMRP. In some embodiments, synthetic nucleic acids could be used as benchmarks to screen and identify other types of small molecule inhibitors that act to block FMRP function.

[0161] Any suitable assay known in the art for identifying inhibitors of RNA-binding proteins can be used to screen compound libraries for small molecules that disrupt the binding of FMRP to its characteristic target mRNA (see, e.g., Roos et al., 2016). The readout involves identifying compounds that, upon binding, release the fluorescence quenching of a fluorophore covalently attached to the protein, the emission of which would otherwise be blocked by the bound target RNA molecule modified to carry the fluorescence quencher. When applied to FMRP, this method is performed as follows and is shown schematically in Figure 10.

[0162] For example, 1) human FMRP protein is produced in human HEK293 cells, purified, and biochemically labeled with the fluorescent reporter fluorescein. 2) sc1 RNA is labeled with a fluorescent quencher molecule, such as Cy3 or BHQ, so that the excitable fluorescence emission of FITC is quenched when sc1 binds to FMRP. 3) FITC-FMRP and sc1-Cy3 / BCG are combined and dispensed into 384-well microwells, after which compounds from a large compound library are added to each well. This assay is best performed in an HTP screening facility, where a robot prepares the microwells containing the protein / RNA complexes, adds coded compounds to each well, and reads the fluorescence emission. 4) Compounds that induce the release of quenched fluorescence are further characterized to verify their ability to inhibit the interaction between FMRP and sc1. 5) Such "leads" are then further characterized to determine binding affinity and kinetic parameters using microscale thermophoresis (MST) measurements and biolayer interferometry (BLI by ForteBio-Octet). Newly identified compounds are further tested in biochemical, structural, cell-based assays, and tumor models to determine whether they inhibit FMRP function and / or whether they tightly bind to the FMRP protein regardless of functional inhibition. Tight binders, whether or not they prove to be functional inhibitors themselves, may serve as building blocks for proteolytic molecules designed to selectively degrade FMRP protein.

[0163] Example 5 Identification of compounds that inhibit FMRP / FMR1 expression Cancer cells expressing high levels of endogenous FMRP protein and mRNA are transfected with an FMRP promoter driving GFP and a ubiquitous promoter driving RFP. A cell-based HTP screen is then performed to score compounds that suppress green fluorescence (FMRP transcription) but not red fluorescence (cell viability). Since true inhibitors of FMR1 transcription should suppress not only the reporter gene but also FMR1 itself, initial hit compounds are filtered by immunostaining endogenously expressed FMRP. A variation is to engineer a cell line with an FMRP promoter driving a fusion gene consisting of FMRP and GFP, which can also be used to score translation inhibitors and protein stability inhibitors. HTP cell-based screening methodologies for identifying transcription inhibitors are being successfully applied (see, e.g., Zhang, 2018; Vuong, 2016).

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Claims

1. An agent for use in the treatment and / or prevention of cancer and / or cancer metastasis in a subject in need thereof, which modulates the expression and / or activity of i) FMRP protein, ii) mRNA encoding FMRP and / or iii) FMR1 gene.

2. The agent of claim 1, wherein the modulation of the expression and / or activity of the FMRP protein comprises a regulatory interaction of the FMRP protein with its mRNA target and / or other proteins.

3. 3. The agent for use according to claim 1 or claim 2, wherein the cancer and / or cancer metastases are inherently resistant or have acquired adaptive resistance to immunotherapy.

4. 4. An agent for use according to any one of claims 1 to 3, which inhibits translation of RNA encoding FMRP.

5. 4. A drug for use according to any one of claims 1 to 3, which inhibits transcription of the FMR1 gene encoding FMRP.

6. 4. An agent for use according to any one of claims 1 to 3, wherein the agent inhibits or impairs binding of the FMRP to a target mRNA or miRNA.

7. 7. The agent for use according to any one of claims 1 to 6, wherein the agent is a compound, a peptide or analogue thereof, an antibody or an antigen-binding fragment of said antibody, an antibody mimetic, or a nucleic acid.

8. The agent of claim 7, wherein the nucleic acid is selected from the group comprising nucleic acids encoding miRNA, siRNA, piRNA, hnRNA, snRNA, sgRNA, CRISPR-based loss-of-function systems, esiRNA, shRNA, and antisense oligonucleotides, or combinations thereof.

9. A plasmid or vector comprising one or more nucleic acids encoding the miRNA, siRNA, piRNA, hnRNA, snRNA, sgRNA, CRISPR-based loss-of-function system, eSiRNA, shRNA, and / or antisense oligonucleotide of claim 7.

10. A host cell comprising one or more nucleic acids encoding the plasmid or vector of claim 9, or the miRNA, siRNA, piRNA, hnRNA, snRNA, sgRNA, CRISPR-based loss-of-function system, esiRNA, shRNA, and / or antisense oligonucleotide of claim 8.

11. The agent of claim 7, wherein the nucleic acid is selected from the group comprising nucleic acids encoding the peptide or an analog thereof, an antibody or an antigen-binding fragment of the antibody, or an antibody mimetic.

12. A plasmid or vector comprising one or more nucleic acids encoding the peptide or analog thereof, antibody or antigen-binding fragment of said antibody, or antibody mimetic of claim 11.

13. 1. A pharmaceutical composition comprising: i) a therapeutically effective amount of an agent that modulates the expression and / or activity of FMRP protein, mRNA encoding FMRP, and / or the FMR1 gene; or ii) A plasmid or vector according to claim 9 or claim 11, or iii) A host cell according to claim 10 or claim 12; a pharmaceutically acceptable carrier or diluent; A pharmaceutical composition comprising:

14. 14. The pharmaceutical composition of claim 13 for use in the treatment and / or prevention of cancer and / or cancer metastasis in a subject in need thereof.

15. 15. The pharmaceutical composition of claim 13 or claim 14, further comprising one or more anti-cancer therapies.

16. 16. The pharmaceutical composition of claim 15, wherein the one or more anti-cancer therapies comprises a therapeutically effective amount of an immune checkpoint inhibitor.

17. 16. The pharmaceutical composition of claim 15, wherein the immune checkpoint inhibitor is selected from the group comprising a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor, or a combination thereof.

18. 18. The pharmaceutical composition of any one of claims 15 to 17, wherein the one or more anti-cancer therapies directed against FMRP are included in combination with a therapeutically effective amount of an anti-tumor vaccine comprising a personalized neoantigen cocktail or other immunostimulatory agent that enhances the anti-tumor immune response.

19. A method for treating and / or preventing cancer and / or cancer metastasis in a subject in need thereof, comprising administering to said subject a pharmaceutical composition according to any one of claims 13 to 18.

20. A method for identifying an agent that modulates the expression and / or activity of i) FMRP protein, ii) mRNA encoding FMRP, and / or iii) FMR1 gene, comprising employing the gene encoding FMRP in an in vivo or in vitro assay to identify said agent.

21. 1. A method for identifying an agent that modulates the expression and / or activity of i) FMRP protein, ii) RNA encoding mFMRP, and / or iii) the FMR1 gene, comprising: (1) providing a sample expressing FMRP; (2) contacting the biological sample with a test agent; (3) determining the level of expression and / or activity of FMRP; (4) comparing the level of expression and / or activity with a control sample that has not been contacted with the test agent; (5) selecting a test agent that reduces the level of expression and / or activity of the FMRP; A method comprising:

22. 22. The method of claim 21, wherein the level of expression and / or activity of FMRP is determined by the agent that inhibits binding of FMRP to its target mRNA, miRNA, or protein.

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