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WO2022061112A1 - Traitement combiné pour le cancer - Google Patents

Traitement combiné pour le cancer Download PDF

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Publication number
WO2022061112A1
WO2022061112A1 PCT/US2021/050870 US2021050870W WO2022061112A1 WO 2022061112 A1 WO2022061112 A1 WO 2022061112A1 US 2021050870 W US2021050870 W US 2021050870W WO 2022061112 A1 WO2022061112 A1 WO 2022061112A1
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Prior art keywords
nucleic acid
mertk
seq
targeted nucleic
radiotherapy
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English (en)
Inventor
James Welsh
Maria Angelica CORTEZ
Yun Hu
Hampartsoum BARSOUMIAN
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/10—X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy
    • A61N2005/1092—Details
    • A61N2005/1098—Enhancing the effect of the particle by an injected agent or implanted device
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy

Definitions

  • aspects of this invention relate to at least the fields of cancer biology, immunology, and medicine.
  • Radiotherapy is known to be effective for local treatment of cancer. Recently, radiotherapy has also been shown to sensitize the tumor microenvironment to immunotherapy treatments. However, radiotherapy can have adverse immunosuppressive effects, which prevent achievement of a systemic response to cancer therapies, such as immunotherapies, at metastatic tumor sites and decrease the efficacy of cancer therapies in treating metastatic cancers. There is a need in the art for methods and compositions for sensitizing such metastatic cancers to radiotherapy or other cancer therapies.
  • aspects of the present disclosure address needs in the art by providing methods and compositions for treating subjects with cancer (e.g., lung cancer) and for sensitizing a subject with cancer to radiotherapy and/or immunotherapy.
  • methods and compositions for treating a subject with cancer comprising providing a triple combination therapy comprising an radiotherapy, a checkpoint inhibitor therapy, and a MER proto-oncogene tyrosine kinase (MerTK)-targeted nucleic acid to the subject, where the subject has been diagnosed with or is suspected of having cancer.
  • the disclosed methods comprise providing the triple combination therapy to a subject who was previously treated for cancer and was determined to be resistant to the previous treatment.
  • the previous cancer treatment comprised a radiotherapy and/or an immunotherapy.
  • Embodiments of the disclosure include methods and compositions for treating a subject having cancer, methods for diagnosing a subject with cancer, methods for prognosing a subject with cancer, methods and compositions for sensitizing a subject with cancer to radiotherapy or immunotherapy, methods for identifying a subject with cancer as a candidate for a combination therapy, and methods and compositions for treating a subject having lung cancer.
  • Methods of the disclosure can include 1, 2, 3, 4, 5, 6, or more of the following steps: providing a radiotherapy to a subject, providing an immunotherapy to a subject, providing a MerTK-targeted nucleic acid to a subject, providing a triple combination therapy to a subject, providing an alternative therapy to a subject, determining a subject to have cancer, providing two or more types of cancer therapy to a subject, identifying a subject as having had resistance to a previous cancer treatment, testing a subject for resistance to previous cancer treatments, and identifying a subject as being a candidate for a triple combination therapy comprising radiotherapy, a checkpoint inhibitor therapy, and a MerTK-targeted nucleic acid. Certain embodiments of the disclosure may exclude one or more of the preceding elements and/or steps.
  • a method for treating a subject for cancer comprising administering to the subject a radiotherapy, a checkpoint inhibitor therapy, and a MerTK-targeted nucleic acid.
  • a method for stimulating an immune-mediated response at a metastatic tumor site comprising administering to the subject a radiotherapy, a checkpoint inhibitor therapy, and a MerTK- targeted nucleic acid.
  • administering the radiotherapy, the checkpoint inhibitor therapy, and the MerTK-targeted nucleic acid decreases radiotherapy-induced immunosuppression of a systemic response to an immunotherapy treatment.
  • administering the radiotherapy, the checkpoint inhibitor therapy, and the MerTK-targeted nucleic acid inhibits primary and secondary tumor growth in the subject.
  • administering the radiotherapy, the checkpoint inhibitor therapy, and the MerTK-targeted nucleic acid shifts tumor-associated macrophage polarization from a protumor M2 phenotype to an anti-tumor Ml phenotype, and macrophage polarization is shifted in secondary tumors.
  • administering the radiotherapy, the checkpoint inhibitor therapy, and the MerTK-targeted nucleic acid increases a population of immune cells in primary and/or secondary tumors, and the population of immune cells comprises CD4+ T- cells, CD8+ T-cells, B-cells, and/or NK cells.
  • the MerTK-targeted nucleic acid is an antisense oligonucleotide, a single- stranded deoxyribonucleic acid molecule, a small interfering RNA, a small hairpin RNA, a viral vector, or a guide RNA.
  • the MerTK- targeted nucleic acid is an antisense oligonucleotide.
  • the MerTK- targeted nucleic acid is a deoxyribonucleic acid (DNA) molecule.
  • the MerTK-targeted nucleic acid is a ribonucleic acid (RNA) molecule.
  • the MerTK-targeted nucleic acid comprises a sequence having at least 70%, at least 71%, at least
  • the MerTK-targeted nucleic acid comprises a sequence having at least 95% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO: 9.
  • the MerTK-targeted nucleic acid comprises a sequence having at least 95% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:
  • the MerTK- targeted nucleic acid comprises a sequence having at least 95% sequence identity with SEQ ID NO:1.
  • the MerTK-targeted nucleic acid comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ
  • the MerTK-targeted nucleic acid comprises SEQ ID NO:1. In specific embodiments, the MerTK-targeted nucleic acid comprises SEQ ID NO:2. In specific embodiments, the MerTK-targeted nucleic acid comprises SEQ ID NO:
  • the MerTK-targeted nucleic acid comprises SEQ ID NO:4. In specific embodiments, the MerTK-targeted nucleic acid comprises SEQ ID NO:5. In specific embodiments, the MerTK-targeted nucleic acid comprises SEQ ID NO:6. In specific embodiments, the MerTK-targeted nucleic acid comprises SEQ ID NO:7. In specific embodiments, the MerTK-targeted nucleic acid comprises SEQ ID NO:8. In specific embodiments, the MerTK-targeted nucleic acid comprises SEQ ID NO:9. [0010] In some embodiments, the MerTK-targeted nucleic acid is configured to bind to a MerTK mRNA. In some embodiments, the MerTK-targeted nucleic acid is configured to reduce expression of a MerTK protein.
  • a single dose of the MerTK-targeted nucleic acid is administered. In some embodiments, multiple doses of the MerTK-targeted nucleic acid are administered. In some embodiments, the MerTK-targeted nucleic acid is administered at a dose of between 1 mg/kg and 5000 mg/kg, or any value or range derivable therein. In some embodiments, the MerTK-targeted nucleic acid is administered intratumorally, intravenously, intramuscularly, intraperitoneally, subcutaneously, intraarticularly, intrasynovially, intrathecally, orally, topically, through inhalation, or through a combination of two or more routes of administration.
  • the checkpoint inhibitor therapy comprises a cytotoxic T- lymphocyte-associated protein 4 (CTLA4) inhibitor, a programmed cell death protein 1 (PD1) inhibitor, a programmed death-ligand 1 (PDL-1) inhibitor, a lymphocyte activation gene-3 (LAG3) inhibitor, or a T-cell immunoglobulin and mucin domain 3 (TIM-3) inhibitor.
  • CTLA4 cytotoxic T- lymphocyte-associated protein 4
  • PD1 programmed cell death protein 1
  • PDL-1 programmed death-ligand 1
  • LAG3 lymphocyte activation gene-3
  • TIM-3 T-cell immunoglobulin and mucin domain 3
  • the checkpoint inhibitor therapy comprises a PD1 inhibitor.
  • the PD1 inhibitor is an anti-PDl antibody.
  • the checkpoint inhibitor therapy comprises a PDL-1 inhibitor.
  • the PDL- 1 inhibitor is an anti-PDL-1 antibody.
  • the checkpoint inhibitor therapy comprises a CTLA4 inhibitor.
  • the CTLA4 inhibitor is an anti-CTLA4 antibody.
  • the checkpoint inhibitor is administered at a dose of between 1 mg/kg and 100 mg/kg, or any range or value derivable therein.
  • the checkpoint inhibitor therapy is administered intratumorally, intravenously, intramuscularly, intraperitoneally, subcutaneously, intraarticularly, intrasynovially, intrathecally, orally, topically, through inhalation, or through a combination of two or more routes of administration.
  • the radiotherapy comprises external radiotherapy, internal radiotherapy, radioimmunotherapy, or intraoperative radiation therapy (IORT).
  • the external radiotherapy comprises three-dimensional conformal radiation therapy (3D-CRT), intensity modulated radiation therapy (IMRT), proton beam therapy, image-guided radiation therapy (IGRT), or stereotactic radiation therapy.
  • the internal radiotherapy comprises interstitial brachytherapy, intracavitary brachytherapy, or intraluminal radiation therapy.
  • the radiotherapy administered to the subject provides irradiation at a dose of between 0.5 Gy and 60 Gy, or any range or value derivable therein. In some embodiments, the radiotherapy is administered in a single dose.
  • the radiotherapy is administered in a fractionated dose over a period of time of not more than one week. In some embodiments, the radiotherapy is delivered in a fractionated dose over a period of time of not more than three days. In some embodiments, the radiotherapy is administered to a primary tumor.
  • the checkpoint inhibitor therapy and the MerTK-targeted nucleic acid are administered substantially simultaneously.
  • the radiotherapy, the checkpoint inhibitor therapy, and the MerTK-targeted antisense oligonucleotide are administered substantially simultaneously.
  • the checkpoint inhibitor therapy and the MerTK-targeted nucleic acid are administered sequentially.
  • the radiotherapy, the checkpoint inhibitor therapy, and the MerTK-targeted nucleic acid are administered sequentially.
  • the MerTK-targeted nucleic acid is administered before administering the radiotherapy.
  • the MerTK-targeted nucleic acid is administered after administering the radiotherapy.
  • a first dose of the MerTK-targeted nucleic acid is administered before administering the radiotherapy and a second dose of the MerTK-targeted nucleic acid is administered after administering the radiotherapy.
  • the method further comprises, prior to administering to the subject the radiotherapy, the checkpoint inhibitor therapy, and the MerTK-targeted nucleic acid, diagnosing the subject with cancer.
  • the subject was previously treated for the cancer.
  • the subject was determined to be resistant to previous treatment.
  • the previous treatment comprised radiotherapy and/or a checkpoint inhibitor therapy.
  • the cancer is colon cancer, prostate cancer, lung cancer, melanoma, or breast cancer.
  • the cancer is lung cancer.
  • the lung cancer is non-small cell lung cancer.
  • the cancer is metastatic cancer.
  • a pharmaceutical composition comprising a nucleic acid having at least 95% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOG, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NOG, or SEQ ID NO: 9, and a pharmaceutically acceptable excipient.
  • the nucleic acid comprises a nucleic acid having at least 95% sequence identity with SEQ ID NO:1.
  • the nucleic acid comprises SEQ ID NO:1, , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO: 9.
  • the nucleic acid comprises SEQ ID NO: 1.
  • the nucleic acid comprises SEQ ID NO:2.
  • the nucleic acid comprises SEQ ID NO:3.
  • the nucleic acid comprises SEQ ID NO:4.
  • the nucleic acid comprises SEQ ID NO:5.
  • the nucleic acid comprises SEQ ID NO:6.
  • the nucleic acid comprises SEQ ID NO:7.
  • the nucleic acid comprises SEQ ID NO:8. In specific embodiments, the nucleic acid comprises SEQ ID NO:9. In some embodiments, the MerTK-targeted nucleic acid is at a concentration of between 1 mg/kg and 5000 mg/kg.
  • the pharmaceutical composition further comprises one or more additional therapeutics.
  • the one or more additional therapeutics comprise a checkpoint inhibitor.
  • the checkpoint inhibitor therapy comprises a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitor, a programmed cell death protein 1 (PD1) inhibitor, a programmed death-ligand 1 (PDL-1) inhibitor, a lymphocyte activation gene-3 (LAG3) inhibitor, or a T-cell immunoglobulin and mucin domain 3 (TIM-3) inhibitor.
  • CTLA4 cytotoxic T-lymphocyte-associated protein 4
  • PD1 inhibitor a programmed cell death protein 1
  • PDL-1 programmed death-ligand 1
  • LAG3 lymphocyte activation gene-3
  • TIM-3 T-cell immunoglobulin and mucin domain 3
  • the checkpoint inhibitor therapy comprises a PD1 inhibitor.
  • the PD1 inhibitor is an anti-PDl antibody.
  • the checkpoint inhibitor therapy comprises a PDL-1 inhibitor.
  • the PDL-1 inhibitor is an anti-PDL-1 antibody.
  • the checkpoint inhibitor therapy comprises a CTLA4 inhibitor.
  • the CTLA4 inhibitor is an anti-CTLA4 antibody.
  • the checkpoint inhibitor is at a concentration of between 1 mg/kg and 100 mg/kg.
  • “Individual, “subject,” and “patient” are used interchangeably and can refer to a human or non-human.
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • “and/or” operates as an inclusive or.
  • compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.
  • any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.
  • FIG. 1 shows a schematic representation of an example treatment regimen for a triple combination therapy for mice with 344SQR two tumor models.
  • FIG. 2 shows average tumor volumes and demonstrates that the combination of XRT+MerTK+anti-PDl antibody improves primary tumor control.
  • IP Intraperitoneal injection
  • IT Intratumoral injection.
  • Error bars represent SEM.
  • FIG. 3 demonstrates that the combination of XRT+MertK+anti-PDl antibody improves abscopal effect.
  • IP Intraperitoneal injection
  • IT Intratumoral injection.
  • P ⁇ 0.05 is considered statistically significant. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, NS, not significant.
  • FIG. 4 shows survival curves and demonstrates that the combination of XRT+MertK+anti-PDl antibody extends survival.
  • FIGS. 5A-5B show individual tumor growth curves.
  • the mice were inoculated with primary tumors (FIG. 5A) and secondary tumors (FIG. 5B) on the right and left legs on days 0 and 4, respectively.
  • the primary tumors were irradiated with 3x12 Gy radiation on day 8, 9, and 10.
  • the mice were intraperitoneally administered with 10 mg/kg aPDl and 50 mg/kg MerTK ASO on the indicated time points in FIG. 1.
  • the tumor volumes were monitored from day 7 and the mice were sacrificed when any dimension of the tumors reached 14 mm.
  • the tumors volumes were compared with two-way ANOVA and expressed as Mean ⁇ SEM.
  • the survival curves were compared with log-rank tests.
  • P values of ⁇ 0.05 indicates statistical significance. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001, NS denotes not significant.
  • FIGS. 6A-6F Triple combination of XRT, MerTK ASO, and anti-CTLA4 achieved a better abscopal effect than the dual combination of XRT, MerTK ASO, and anti-CTLA4.
  • FIG. 6A Treatment scheme for mice with 344SQR two tumor models. Average primary (FIG. 6B) and secondary (FIG. 6C) tumor volumes and survival curves (FIG. 6D). Individual tumor growth curves are also shown. The mice were inoculated with primary tumors (FIG. 6E) and secondary tumors (FIG. 6F) on the right and left legs on days 0 and 4, respectively. The primary tumors were irradiated with 3xl2Gy radiation on day 8, 9, and 10.
  • mice were intraperitoneally administered with 2.5 mg/kg anti-CTLA4 and 50 mg/kg MerTK ASO on the indicated time points in FIG. 6A.
  • the tumor volumes were monitored from day 7 and the mice were sacrificed when any dimension of the tumors reached 14 mm.
  • the tumors volumes were compared with two-way ANOVA and expressed as mean ⁇ SEM.
  • the survival curves were compared with log-rank tests. P values of ⁇ 0.05 indicates statistical significance. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001, NS denotes not significant.
  • FIG. 7A-7H MerTK ASO reshapes the population of immune cells in TME in favor of antitumor immune response (FIG. 7A).
  • the percentages of MerTK + tumor-associated macrophages (TAMs). (FIG. 7B). The MerTK expression level in TAMS. (FIG. 7C). Representative FACS images of MerTK + macrophages in the primary tumors. (FIG. 7D). Representative FACS images of MerTK + macrophages in the secondary tumors. (FIG. 7E). M1/M2 ratio. (FIG. 7F). CD8 + T-cell/CD45 + cell ratio. (FIG. 7G). CD4 + T-cell/CD45 + cell ratio. (FIG. 7H). The percentages of GrB + CD8 + T-cells out of total CD8 + T-cells.
  • mice were treated with different combinations of XRT, MerTK ASO, aPDl, and aCTEA4, as indicated in FIG. 1 and FIG. 6A, and were sacrificed on day 21. Both the primary and the secondary tumors were harvested and stained with aCD45-PerCP-Cy5.5, aCD4- PE/Dazzle594, aCD8-FITC, aGrB-Pacific Blue, aGrl-BV510, aCDl lb-APC Fire750, aF4/80-Alexa Fluor 700, aCD38-PE-Cy7, aCD206-PE, aMertK-APC. All the statistics were compared with two-tailed t tests and expressed as mean value ⁇ SEM.
  • RNA extracted from the primary tumors was analyzed with a nCounter PanCancer Immune Profiling Panel. All the statistics were compared with two-tailed t tests and expressed as mean value ⁇ SEM. P values of ⁇ 0.05 indicates statistical significance. *P ⁇ 0.05, **P ⁇ 0.01, NS denotes significant.
  • FIGS. 9A-9C MerTK ASO modulates the expression of immune-related genes in the secondary tumors.
  • FIG. 9A Scores of various immune pathways in the primary tumors of the mice treated with XRT+MerTK, XRT+aPDl, XRT+aCTEA4, RPM, and RCM.
  • MerTK ASO induced changes in expression of genes in the adaptive, innate, macrophage function, T- cell function, DC function, and NK cell function.
  • RNA extracted from the primary tumors was analyzed with a nCounter PanCancer Immune Profiling Panel. All statistics were done using two-tailed t tests and expressed as mean value + SEM. P values of ⁇ 0.05 indicates statistical significance. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, NS denotes not significant.
  • FIG. 10 MerTK ASO causes no retinal toxicity. Eyes from mice were harvested on day 21 and stained with H H&E and toluidine blue.
  • FIGS. 11A-11F Combination of XRT, MerTK ASO, and anti-PDl delays the growth of both the primary and the secondary tumors (FIG. 11A).
  • Primary tumors were irradiated with 3x12 Gy radiation on days 8, 9, and 10.
  • mice were intraperitoneally administered with 10 mg/kg anti-PDl and 50 mg/kg MerTK ASO on the indicated time points in supplemental FIG. 1.
  • the tumor volumes were monitored from day 7 and the mice were sacrificed when any dimension of the tumors reached 14 mm.
  • the tumors volumes were compared with two-way ANOVA and expressed as mean ⁇ SEM.
  • the survival curves were compared with log-rank tests. P values of ⁇ 0.05 indicates statistical significance. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, NS denotes not significant.
  • FIG. 12 Nanostring scores of various immune cells in the primary tumors.
  • the total RNA extracted from the primary tumors was analyzed with a nCounter PanCancer Immune Profiling Panel. All the statistics were expressed as mean value ⁇ SEM.
  • FIG. 13 Nanostring scores of various immune cells in the secondary tumors.
  • the total RNA extracted from the primary tumors was analyzed with an nCounter PanCancer Immune Profiling Panel. All statistics were expressed as mean value ⁇ SEM.
  • FIG. 14 MerTK ASO significantly changed the activity of various pathways in the secondary tumor when added to XRT+ocPDl and XRT+ocCTLA4.
  • the present disclosure is based, at least in part, on the surprising discovery that cancer associated with metastases resistant to immunotherapy is responsive to a triple combination therapy comprising a radiotherapy, a checkpoint inhibitor therapy, and a MER proto-oncogene tyrosine kinase (MerTK) -targeted nucleic acid. Further, administering a radiotherapy, a checkpoint inhibitor therapy, and a MerTK-targeted nucleic acid to a subject was surprisingly found to decrease radiotherapy-induced immunosuppression of a systemic response to an immunotherapy treatment and to inhibit primary and secondary tumor growth in the subject.
  • MerTK proto-oncogene tyrosine kinase
  • a critical receptor that mediates the clearance of apoptotic cells by macrophages is MER proto-oncogene tyrosine kinase (MerTK).
  • MerTK proto-oncogene tyrosine kinase
  • TYRO3 and AXL MER proto-oncogene tyrosine kinase
  • MerTK is a member of a receptor tyrosine kinase family (Rothlin et al., 2015).
  • MerTK is overexpressed or ectopically expressed in a wide variety of cancers, including leukemia, non-small cell lung cancer, and glioblastoma, and can potentially activate several canonical oncogenic signaling pathways (Cummings et al., 2013).
  • MerTK signaling promotes the secretion of anti-inflammatory cytokines, such as TGFp, hepatocyte growth factor (HGF), and IL- 10, resulting in pro-tumor M2-polarized macrophages in the tumor microenvironment (TME) (Huelse et al., 2020; Myers et al., 2019). Therefore, MerTK on macrophages has been identified as a potential therapeutic target for cancer treatment (Crittenden et al., 2016; Myers et al., 2019).
  • UNC569 a small molecule MerTK inhibitor, also displayed potent antitumor efficacy both in vitro and in vivo (Christoph et al., 2013; Liu et al., 2012).
  • ASO MerTK specific anti-sense oligonucleotide
  • a triple combination therapy comprising a radiotherapy, a checkpoint inhibitor therapy, and a MerTK-targeted ASO nucleic acid
  • cancer for example, metastatic cancer
  • the addition of the MerTK ASO to the combination of radiotherapy and checkpoint inhibitor (CPI) was observed to reliably induce an abscopal effect, with substantial reductions in secondary tumor growth and coordinate improvements in animal survival.
  • mice treated with XRT+CPI alone - specifically, XRT+aPDl - indicates that the MerTK ASO was able to convert otherwise aPDl resistant tumors to aPDl -sensitive tumors.
  • XRT, MerTK ASO, and CPIs promoted Ml macrophage polarization, facilitated the infiltration of effector immune cells, upregulated the activities of antitumor immune pathways by modulating the expression of immune-related genes, and promoted the activation of CD8 + T-cells in the unirradiated tumors, all of which resulted in an improved abscopal effect in an aPDl resistant tumor.
  • a radiotherapy comprising administering a radiotherapy, a checkpoint inhibitor therapy, and a MerTK-targeted nucleic acid to a subject having or suspected or having cancer.
  • the cancer is metastatic cancer.
  • the subject has or has previously had resistance to radiotherapy or immunotherapy.
  • Further aspects disclose methods for stratifying cancer patients based on a history of resistance to radiotherapy or immunotherapy. For example, embodiments are directed to methods for identifying a subject as being a candidate for a triple combination therapy comprising radiotherapy, a checkpoint inhibitor therapy, and a MerTK-targeted nucleic acid by identifying the subject as having or having previously had resistance to radiotherapy or immunotherapy.
  • compositions of the disclosure may be used for in vivo, in vitro, or ex vivo administration.
  • the route of administration of the composition may be, for example, intratumoral, intravenous, intramuscular, intraperitoneal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, through inhalation, or through a combination of two or more routes of administration.
  • the disclosed methods comprise administering a cancer therapy to a subject or patient.
  • the cancer therapy may be chosen based on the expression level measurements, alone or in combination with the clinical risk score calculated for the subject.
  • the cancer therapy comprises a local cancer therapy.
  • the cancer therapy excludes a systemic cancer therapy.
  • the cancer therapy excludes a local therapy.
  • the cancer therapy comprises a local cancer therapy without the administration of a system cancer therapy.
  • the cancer therapy comprises an immunotherapy, which may be a checkpoint inhibitor therapy. Any of these cancer therapies may also be excluded. Combinations of these therapies may also be administered.
  • cancer may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer.
  • the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;
  • the cancer is lung cancer.
  • the cancer is non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • the cancer is leukemia.
  • the cancer is glioblastoma.
  • the cancer is colon cancer.
  • the cancer is prostate cancer.
  • the cancer is melanoma.
  • the cancer is breast cancer.
  • the disclosed methods comprise administration of a MER proto-oncogene tyrosine kinase (MerTK)-targeted nucleic acid.
  • MerTK is a member of the TYRO3/AXL/MER (TAM) receptor kinase family and encodes a transmembrane protein with two fibronectin type-III domains, two Ig-like C2-type (immunoglobulin-like) domains, and one tyrosine kinase domain.
  • TAM TYRO3/AXL/MER
  • MerTK is expressed in monocytes/macrophages, dendritic cells, NK cells, NKT cells, megakaryocytes, and platelets. High levels of MerTK expression are also detected in ovary, prostate, testis, lung, retina, and kidney. Lower levels of MerTK are found in heart, brain, and skeletal muscle.
  • a “MerTK-targeted nucleic acid” describes any nucleic acid molecule capable of reducing or eliminating expression of a MerTK protein in a cell.
  • a MerTK-targeted nucleic acid is a nucleic acid capable of binding to a region of a MerTK messenger RNA.
  • a MerTK-targeted nucleic acid is a nucleic acid capable of binding to a region of a MERTK gene or a complement thereof.
  • Examples of MerTK-targeted nucleic acids of the present disclosure include, but are not limited to, an antisense oligonucleotide, a single-stranded deoxyribonucleic acid molecule, a small interfering RNA, a small hairpin RNA, a viral vector, or a guide RNA.
  • the MerTK-targeted nucleic acid is an antisense oligonucleotide.
  • Oligonucleotide refers to a plurality of joined nucleotide units formed in a specific sequence from naturally occurring bases and pentofuranosyl groups joined through a sugar group by native phosphodiester bonds. This term refers to both naturally occurring and synthetic species formed from naturally occurring subunits.
  • the oligonucleotide may be a modified oligonucleotide that has non-naturally occurring portions. Modified oligonucleotide can have altered sugar moieties, altered base moieties or altered inter-sugar linkages.
  • oligomers is intended to encompass oligonucleotides, oligonucleotide analogs or oligonucleosides. Thus, in speaking of “oligomers” reference is made to a series of nucleosides or nucleoside analogs that are joined via either natural phosphodiester bonds or other linkages, including the four atom linkers. Although the linkage generally is from the 3’ carbon of one nucleoside to the 5’ carbon of a second nucleoside, the term “oligomer” can also include other linkages such as 2’- 5’ linkages.
  • Modified oligonucleotides can include modifications that increase nuclease resistance, improve binding affinity, and/or improve binding specificity. For example, when the sugar portion of a nucleoside or nucleotide is replaced by a carbocyclic moiety, it is no longer a sugar. Moreover, when other substitutions, such a substitution for the inter-sugar phosphodiester linkage are made, the resulting material is no longer a true nucleic acid species. All such compounds are considered to be analogs.
  • the modified oligonucleotides may exhibit increased chemical and/or enzymatic stability relative to their naturally occurring counterparts.
  • Extracellular and intracellular nucleases generally do not recognize and therefore do not bind to the backbone-modified compounds.
  • the lack of a negatively charged backbone may facilitate cellular penetration.
  • the modified intemucleoside linkages are intended to replace naturally-occurring phosphodiester-5’ -methylene linkages with four atom linking groups to confer nuclease resistance and enhanced cellular uptake to the resulting compound.
  • Preferred linkages have structure CH 2 -RA — NRi CH 2 , CH 2 — NRi — RA -CH 2 , RA — NRi -CH 2 -, CH 2 -CH 2 -NRi — RA, CH 2 -CH 2 -RA -NRi, or NRi -RA -CH 2 -CH 2 where RA is O or NR 2 .
  • Modifications may be achieved using solid supports which may be manually manipulated or used in conjunction with a DNA synthesizer using methodology commonly known to those skilled in DNA synthesizer art. Generally, the procedure involves functionalizing the sugar moieties of two nucleosides which will be adjacent to one another in the selected sequence. In a 5’ to 3’ sense, an “upstream” synthon such as structure H is modified at its terminal 3’ site, while a “downstream” synthon such as structure Hl is modified at its terminal 5’ site.
  • Antisense oligonucleotides of the disclosure may be equal to any one of, at least any one of, at most any one of, or between any two of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides (or any derivable range therein).
  • oligonucleotides of the disclosure include a flanking sequence. Several types of flanking sequences may be used. In some embodiments, flanking sequences are used to modify the binding of a protein to said molecule or oligonucleotide, or to modify a thermodynamic property of the oligonucleotide, or to modify target RNA binding affinity.
  • an oligonucleotide of the present disclosure comprises a nucleotide-based or nucleotide or an antisense oligonucleotide sequence of between 3 and 30 nucleotides or bases, between 5 and 25 nucleotides, or between 10 and 20 nucleotides, such as 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, or 20 nucleotides.
  • an oligonucleotide of the disclosure comprises a sequence having equal to any one of, at least any one of, at most any one of, or between any two of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with AGTGATATAATGGTCA (SEQ ID NO:1), or any value derivable therein.
  • an oligonucleotide of the disclosure comprises SEQ ID NO:1.
  • an oligonucleotide of the disclosure comprises a sequence having equal to any one of, at least any one of, at most any one of, or between any two of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with GTATTATTATCATGGG (SEQ ID NO:2), or any value derivable therein.
  • an oligonucleotide of the disclosure comprises SEQ ID NO:2.
  • an oligonucleotide of the disclosure comprises a sequence having equal to any one of, at least any one of, at most any one of, or between any two of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with GCAATATCAGTTTCCT (SEQ ID NOG), or any value derivable therein.
  • an oligonucleotide of the disclosure comprises SEQ ID NOG.
  • an oligonucleotide of the disclosure comprises a sequence having equal to any one of, at least any one of, at most any one of, or between any two of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with GTTATTAGTTATCTTA (SEQ ID NOG), or any value derivable therein.
  • an oligonucleotide of the disclosure comprises SEQ ID NOG.
  • an oligonucleotide of the disclosure comprises a sequence having equal to any one of, at least any one of, at most any one of, or between any two of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with GATATCAAAAGCTCAA (SEQ ID NOG), or any value derivable therein.
  • an oligonucleotide of the disclosure comprises SEQ ID NOG.
  • an oligonucleotide of the disclosure comprises a sequence having equal to any one of, at least any one of, at most any one of, or between any two of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with CCCATATATACCTTCC (SEQ ID NOG), or any value derivable therein.
  • an oligonucleotide of the disclosure comprises SEQ ID NOG.
  • an oligonucleotide of the disclosure comprises a sequence having equal to any one of, at least any one of, at most any one of, or between any two of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with GAATATTGATGGAGCA (SEQ ID NOG), or any value derivable therein.
  • an oligonucleotide of the disclosure comprises SEQ ID NOG.
  • an oligonucleotide of the disclosure comprises a sequence having equal to any one of, at least any one of, at most any one of, or between any two of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with TGTGATTAAACGCAAC (SEQ ID NOG), or any value derivable therein.
  • an oligonucleotide of the disclosure comprises SEQ ID NOG.
  • an oligonucleotide of the disclosure comprises a sequence having equal to any one of, at least any one of, at most any one of, or between any two of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with GGTTAATTTGTTTGTC (SEQ ID NO:9), or any value derivable therein.
  • an oligonucleotide of the disclosure comprises SEQ ID NO:9.
  • a nucleotide sequence of an oligonucleotide of the disclosure may contain a RNA residue, a DNA residue, a nucleotide analogue or equivalent as will be further detailed herein.
  • the oligonucleotide comprises at least one residue comprising a modified base, and/or a modified backbone, and/or a non-natural intemucleoside linkage, or a combination of these modifications.
  • the oligonucleotide comprises a modified backbone.
  • backbones are provided by morpholino backbones, carbamate backbones, siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl backbones, riboacetyl backbones, alkene containing backbones, sulfamate, sulfonate and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide backbones.
  • Phosphorodiamidate morpholino oligomers are modified backbone oligonucleotides that have previously been investigated as antisense agents.
  • Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six membered ring and the phosphodiester linkage is replaced by a phosphorodiamidate linkage.
  • Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by arresting translation or interfering with pre-mRNA splicing rather than by activating RNase H.
  • Morpholino oligonucleotides have been successfully delivered to tissue culture cells by methods that physically disrupt the cell membrane, and one study comparing several of these methods found that scrape loading was the most efficient method of delivery; however, because the morpholino backbone is uncharged, cationic lipids are not effective mediators of morpholino oligonucleotide uptake in cells.
  • the modified oligonucleotide comprises a Peptide Nucleic Acid (PNA), having a modified polyamide backbone (Nielsen, et al. (1991) Science 254, 1497- 1500). PNA-based molecules are true mimics of DNA molecules in terms of base-pair recognition.
  • the backbone of the PNA is composed of N-(2-aminoethyl)-glycine units linked by peptide bonds, wherein the nucleobases are linked to the backbone by methylene carbonyl bonds.
  • An alternative backbone comprises a one-carbon extended pyrrolidine PNA monomer (Govindaraju and Kumar (2005) Chem. Commun, 495-497).
  • PNA-RNA hybrids are usually more stable than RNA-RNA or RNA-DNA hybrids, respectively (Egholm et al (1993) Nature 365, 566- 568).
  • the modified oligonucleotide comprises a morpholino nucleotide analog or equivalent, in which the ribose or deoxyribose sugar is replaced by a 6- membered morpholino ring.
  • the modified oligonucleotide comprises phosphorodiamidate morpholino oligomer (PMO), in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring, and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non-ionic phosphorodiamidate linkage.
  • PMO phosphorodiamidate morpholino oligomer
  • the modified oligonucleotide comprises a substitution of at least one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly destabilizes base-pairing but adds significant resistance to nuclease degradation.
  • the modified oligonucleotide comprises phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H- phosphonate, methyl and other alkyl phosphonate including 3 '-alkylene phosphonate, 5'- alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3'- amino phosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate.
  • the modified oligonucleotide comprises one or more sugar moieties that are mono- or disubstituted at the 2', 3' and/or 5' position such as a —OH; — F; substituted or unsubstituted, linear or branched lower (C 1-C 10) alkyl, alkenyl, alkynyl, alkaryl, allyl, aryl, or aralkyl, that may be interrupted by one or more heteroatoms; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; O-, S-, or N-allyl; O-alkyl-O-alkyl, -methoxy, - aminopropoxy; aminoxy, methoxyethoxy; -dimethylaminooxyethoxy; and dimethylaminoethoxyethoxy.
  • a —OH — F
  • the sugar moiety can be a pyranose or derivative thereof, or a deoxypyranose or derivative thereof, preferably a ribose or a derivative thereof, or deoxyribose or derivative thereof.
  • the modified oligonucleotide comprises Locked Nucleic Acid (LNA), in which the 2'-carbon atom is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety.
  • the LNA comprises 2'-O,4'-C-ethylene-bridged nucleic acid (Morita et al. 2001. Nucleic Acid Res Supplement No. 1: 241-242).
  • an antisense oligonucleotide of the invention has at least two different types of analogues or equivalents.
  • the modified oligonucleotide comprises a 2'-O-alkyl phosphorothioate antisense oligonucleotide, such as 2'-O-methyl modified ribose (RNA), 2'- O-ethyl modified ribose, 2'-O-propyl modified ribose, and/or substituted derivatives of these modifications such as halogenated derivatives.
  • the modified oligonucleotide comprises a 2'-O-methyl phosphorothioate ribose.
  • a functional equivalent of a molecule of the disclosure may be defined as an oligonucleotide as defined herein wherein an activity of said functional equivalent is retained to at least some extent.
  • an activity of said functional equivalent is reducing or eliminating MerTK expression in a cell.
  • an activity of said functional equivalent comprises an ability to bind to a MerTK mRNA. Said activity of said functional equivalent therefore may be assessed by detection of binding to MerTK mRNA and/or detection of a reduction or elimination of MerTK expression in a cell.
  • An antisense oligonucleotide can be linked to a moiety that enhances uptake of the antisense oligonucleotide in cells.
  • moieties are cholesterols, carbohydrates, vitamins, biotin, lipids, phospholipids, cell-penetrating peptides including but not limited to antennapedia, TAT, transportan and positively charged amino acids such as oligoarginine, poly-arginine, oligolysine or polylysine, antigen-binding domains such as provided by an antibody, a Fab fragment of an antibody, or a single chain antigen binding domain such as a cameloid single domain antigen-binding domain.
  • the oligonucleotide comprises a peptide-linked PMO.
  • a radiotherapy such as ionizing radiation
  • ionizing radiation means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons).
  • ionizing radiation is an x-radiation.
  • Means for delivering x-radiation to a target tissue or cell are well known in the art.
  • the radiotherapy can comprise external radiotherapy, internal radiotherapy, radioimmunotherapy, or intraoperative radiation therapy (IORT).
  • the external radiotherapy comprises three-dimensional conformal radiation therapy (3D-CRT), intensity modulated radiation therapy (IMRT), proton beam therapy, image-guided radiation therapy (IGRT), or stereotactic radiation therapy.
  • the internal radiotherapy comprises interstitial brachytherapy, intracavitary brachytherapy, or intraluminal radiation therapy.
  • the radiotherapy is administered to a primary tumor.
  • the amount of ionizing radiation is greater than 20 Gy and is administered in one dose. In some embodiments, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some embodiments, the amount of ionizing radiation is equal to any one of, at least any one of, at most any one of, about any one of, or between any two of 0.5, 1, 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 Gy (or any derivable range therein).
  • the ionizing radiation is administered in equal to any one of, at least any one of, at most any one of, about any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses (or any derivable range therein).
  • the does may be equal to any one of, at least any one of, at most any one of, about any one of, or between any two of 1, 4, 8, 12, or 24 hours or 1, 2, 3, 4, 5, 6, 7, or 8 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks apart, or any derivable range therein.
  • the amount of radiotherapy administered to a subject may be presented as a total dose of radiotherapy, which is then administered in fractionated doses.
  • the total dose is 50 Gy administered in 10 fractionated doses of 5 Gy each.
  • the total dose is 50-90 Gy, administered in 20-60 fractionated doses of 2-3 Gy each.
  • the total dose of radiation is equal to any one of, at least any one of, at most any one of, about any one of, or between any two of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,
  • the total dose is administered in fractionated doses of equal to any one of, at least any one of, at most any one of, about any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any derivable range therein).
  • fractionated doses are administered per day.
  • equal to any one of, at least any one of, at most any one of, about any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any derivable range therein) fractionated doses are administered per week.
  • the methods comprise administration of a cancer immunotherapy.
  • Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer.
  • Immunotherapies can be categorized as active, passive or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor-associated antigens (TAAs); they are often proteins or other macromolecules (e.g. carbohydrates).
  • TAAs tumor-associated antigens
  • Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines.
  • Various immunotherapies are known in the art, and examples are described below.
  • Embodiments of the disclosure may include administration of immune checkpoint inhibitors, examples of which are further described below.
  • checkpoint inhibitor therapy also “immune checkpoint blockade therapy”, “immune checkpoint therapy”, “ICT,” “checkpoint blockade immunotherapy,” or “CBI”
  • ICT immune checkpoint therapy
  • CBI checkpoint blockade immunotherapy
  • PD-1 can act in the tumor microenvironment where T-cells encounter an infection or tumor. Activated T-cells upregulate PD- 1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T-cells in the periphery and prevent excessive damage to the tissues during an immune response. Inhibitors of the disclosure may block one or more functions of PD-1 and/or PDL1 activity.
  • Alternative names for “PD-1” include CD279 and SLEB2.
  • Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H.
  • Alternative names for “PDL2” include B7- DC, Btdc, and CD273.
  • PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.
  • the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners.
  • the PD-1 ligand binding partners are PDL1 and/or PDL2.
  • a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners.
  • PDL1 binding partners are PD-1 and/or B7-1.
  • the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners.
  • a PDL2 binding partner is PD-1.
  • the inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference.
  • Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U.S. Patent Application Nos. US2014/0294898, US 2014/022021, and US2011/0008369, all incorporated herein by reference.
  • the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody).
  • the anti-PD- 1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab.
  • the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g. , an Fc region of an immunoglobulin sequence).
  • the PDL1 inhibitor comprises AMP- 224.
  • Nivolumab also known as MDX-1106-04, MDX- 1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W 02006/121168.
  • Pembrolizumab also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009/114335.
  • Pidilizumab also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in W02009/101611.
  • AMP-224 also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in W02010/027827 and WO2011/066342.
  • Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.
  • the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof.
  • the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7.
  • the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another embodiment, the antibody competes for binding with and/or binds to the same epitope on PD-1, PDL1, or PDL2 as the above- mentioned antibodies.
  • the antibody has equal to any one of, at least any one of, at most any one of, about any one of, or between any two of 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.
  • CTLA-4 cytotoxic T-lymphocyte-associated protein 4
  • CD152 cytotoxic T-lymphocyte-associated protein 4
  • Genbank accession number L15006 CTLA-4 is found on the surface of T-cells and acts as an “off’ switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells.
  • CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T-cells and transmits an inhibitory signal to T-cells.
  • CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells.
  • CTLA-4 transmits an inhibitory signal to T-cells, whereas CD28 transmits a stimulatory signal.
  • Intracellular CTLA- 4 is also found in regulatory T-cells and may be important to their function.
  • T-cell activation through the T-cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.
  • Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and/or B7-2 activity.
  • the inhibitor blocks the CTLA-4 and B7-1 interaction.
  • the inhibitor blocks the CTLA-4 and B7-2 interaction.
  • the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • an anti-CTLA-4 antibody e.g., a human antibody, a humanized antibody, or a chimeric antibody
  • an antigen binding fragment thereof e.g., an immunoadhesin, a fusion protein, or oligopeptide.
  • Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art.
  • art recognized anti-CTLA-4 antibodies can be used.
  • the anti- CTLA-4 antibodies disclosed in: US 8,119,129, WO 01/14424, WO 98/42752; WO 00/37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein.
  • the teachings of each of the aforementioned publications are hereby incorporated by reference.
  • CTLA-4 antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used.
  • a humanized CTLA-4 antibody is described in International Patent Application No. WO200 1/014424, W02000/037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.
  • a further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WO 01/14424).
  • the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another embodiment, the antibody competes for binding with and/or binds to the same epitope on PD-1, B7-1, or B7-2 as the above- mentioned antibodies.
  • the antibody has equal to any one of, at least any one of, at most any one of, about any one of, or between any two of 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.
  • LAG3 lymphocyte-activation gene 3
  • CD223 lymphocyte activating 3
  • LAG3 is a member of the immunoglobulin superfamily that is found on the surface of activated T-cells, natural killer cells, B-cells, and plasmacytoid dendritic cells.
  • LAG3’s main ligand is MHC class II, and it negatively regulates cellular proliferation, activation, and homeostasis of T-cells, in a similar fashion to CTLA-4 and PD-1, and has been reported to play a role in Treg suppressive function.
  • LAG3 also helps maintain CD8+ T-cells in a tolerogenic state and, working with PD-1, helps maintain CD8 exhaustion during chronic viral infection.
  • LAG3 is also known to be involved in the maturation and activation of dendritic cells. Inhibitors of the disclosure may block one or more functions of LAG3 activity.
  • the immune checkpoint inhibitor is an anti-LAG3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • an anti-LAG3 antibody e.g., a human antibody, a humanized antibody, or a chimeric antibody
  • an antigen binding fragment thereof e.g., an immunoadhesin, a fusion protein, or oligopeptide.
  • Anti-human-LAG3 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art.
  • art recognized anti-LAG3 antibodies can be used.
  • the anti-LAG3 antibodies can include: GSK2837781, IMP321, FS-118, Sym022, TSR-033, MGD013, BI754111, AVA-017, or GSK2831781.
  • the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-LAG3 antibody. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of an anti-LAG3 antibody, and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-LAG3 antibody. In another embodiment, the antibody has equal to any one of, at least any one of, at most any one of, about any one of, or between any two of 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies. d. TIM-3
  • TIM-3 T-cell immunoglobulin and mucin-domain containing-3
  • HAVCR2 hepatitis A virus cellular receptor 2
  • CD366 CD366
  • the complete mRNA sequence of human TIM-3 has the Genbank accession number NM_032782.
  • TIM-3 is found on the surface IFNy- producing CD4+ Thl and CD8+ Tel cells.
  • the extracellular region of TIM-3 consists of a membrane distal single variable immunoglobulin domain (IgV) and a glycosylated mucin domain of variable length located closer to the membrane.
  • TIM-3 is an immune checkpoint and, together with other inhibitory receptors including PD-1 and LAG3, it mediates the T-cell exhaustion.
  • TIM-3 has also been shown as a CD4+ Thl -specific cell surface protein that regulates macrophage activation.
  • Inhibitors of the disclosure may block one or more functions of TIM- 3 activity.
  • the immune checkpoint inhibitor is an anti-TIM-3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • an anti-TIM-3 antibody e.g., a human antibody, a humanized antibody, or a chimeric antibody
  • an antigen binding fragment thereof e.g., an immunoadhesin, a fusion protein, or oligopeptide.
  • Anti-human-TIM-3 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art.
  • art recognized anti-TIM-3 antibodies can be used.
  • anti-TIM-3 antibodies including: MBG453, TSR-022 (also known as Cobolimab), and LY3321367 can be used in the methods disclosed herein.
  • MBG453, TSR-022 also known as Cobolimab
  • LY3321367 can be used in the methods disclosed herein.
  • These and other anti-TIM-3 antibodies useful in the claimed invention can be found in, for example: US 9,605,070, US 8,841,418, US2015/0218274, and US 2016/0200815.
  • the teachings of each of the aforementioned publications are hereby incorporated by reference.
  • Antibodies that compete with any of these art-recognized antibodies for binding to LAG3 also can be used.
  • the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-TIM-3 antibody. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of an anti-TIM-3 antibody, and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-TIM-3 antibody. In another embodiment, the antibody has equal to any one of, at least any one of, at most any one of, about any one of, or between any two of 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.
  • the immunotherapy comprises an inhibitor of a costimulatory molecule.
  • the inhibitor comprises an inhibitor of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, 0X40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof.
  • Inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.
  • Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen.
  • Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment they aid cancer antigen targeting.
  • APCs antigen presenting cells
  • One example of cellular cancer therapy based on dendritic cells is sipuleucel-T.
  • One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses.
  • adjuvants include proteins or other chemicals that attract and/or activate dendritic cells, such as granulocyte macrophage colony- stimulating factor (GM-CSF).
  • Dendritic cells can also be activated in vivo by making tumor cells express GM- CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF. [0112] Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body. The dendritic cells are activated in the presence of tumor antigens, which may be a single tumor- specific peptide/protein or a tumor cell lysate (a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.
  • tumor antigens which may be a single tumor- specific peptide/protein or a tumor cell lysate (a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.
  • Dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.
  • Chimeric antigen receptors are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T-cell. The receptors are called chimeric because they are fused of parts from different sources.
  • CAR-T-cell therapy refers to a treatment that uses such transformed cells for cancer therapy.
  • CAR-T-cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions.
  • the general premise of CAR-T-cells is to artificially generate T-cells targeted to markers found on cancer cells.
  • scientists can remove T-cells from a person, genetically alter them, and put them back into the patient for them to attack the cancer cells.
  • CAR-T-cells create a link between an extracellular ligand recognition domain to an intracellular signaling molecule which in turn activates T-cells.
  • the extracellular ligand recognition domain is usually a single-chain variable fragment (scFv).
  • scFv single-chain variable fragment
  • Example CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtagene ciloleucel (Yescarta). 5. Cytokine therapy
  • Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immune responses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins.
  • Interferons are produced by the immune system. They are usually involved in antiviral response, but also have use for cancer. They fall in three groups: type I (IFNa and IFNP), type II (IFNy) and type III (IFNk).
  • Interleukins have an array of immune system effects.
  • IL-2 is an example interleukin cytokine therapy.
  • Adoptive T-cell therapy is a form of passive immunization by the transfusion of T- cells (adoptive cell transfer). They are found in blood and tissue and usually activate when they find foreign pathogens. Specifically they activate when the T-cell's surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These can be either infected cells, or antigen presenting cells (APCs). They are found in normal tissue and in tumor tissue, where they are known as tumor infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumor death.
  • APCs antigen presenting cells
  • T-cells specific to a tumor antigen can be removed from a tumor sample (TILs) or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the results reinfused. Activation can take place through gene therapy, or by exposing the T-cells to tumor antigens.
  • TILs tumor sample
  • Activation can take place through gene therapy, or by exposing the T-cells to tumor antigens.
  • a cancer treatment may exclude any of the cancer treatments described herein.
  • embodiments of the disclosure include patients that have been previously treated for a therapy described herein, are currently being treated for a therapy described herein, or have not been treated for a therapy described herein.
  • the patient is one that has been determined to be resistant to a therapy described herein.
  • the patient is one that has been determined to be sensitive to a therapy described herein.
  • the additional therapy comprises an oncolytic virus.
  • An oncolytic virus is a virus that preferentially infects and kills cancer cells. As the infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumor. Oncolytic viruses are thought not only to cause direct destruction of the tumor cells, but also to stimulate host anti-tumor immune responses for long-term immunotherapy
  • the additional therapy comprises polysaccharides.
  • Certain compounds found in mushrooms primarily polysaccharides, can up-regulate the immune system and may have anti-cancer properties.
  • beta-glucans such as lentinan have been shown in laboratory studies to stimulate macrophage, NK cells, T-cells and immune system cytokines and have been investigated in clinical trials as immunologic adjuvants.
  • the additional therapy comprises neoantigen administration.
  • Many tumors express mutations. These mutations potentially create new targetable antigens (neoantigens) for use in T-cell immunotherapy.
  • the presence of CD8+ T-cells in cancer lesions, as identified using RNA sequencing data, is higher in tumors with a high mutational burden.
  • the level of transcripts associated with cytolytic activity of natural killer cells and T- cells positively correlates with mutational load in many human tumors.
  • the additional therapy comprises a chemotherapy.
  • chemotherapeutic agents include (a) Alkylating Agents, such as nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine), (b) Antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and
  • nitrogen mustards e.g.
  • Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes such as, for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, with efficacious doses used in clinical applications including about 15 mg/m 2 to about 20 mg/m 2 for 5 days every three weeks for a total of three courses being contemplated in certain embodiments.
  • the amount of cisplatin delivered to the cell and/or subject in conjunction with the construct comprising an Egr-1 promoter operatively linked to a polynucleotide encoding the therapeutic polypeptide is less than the amount that would be delivered when using cisplatin alone.
  • chemotherapeutic agents include antimicro tubule agents, e.g., Paclitaxel (“Taxol”) and doxorubicin hydrochloride (“doxorubicin”).
  • Paclitaxel e.g., Paclitaxel
  • doxorubicin hydrochloride doxorubicin hydrochloride
  • Doxorubicin is absorbed poorly and is preferably administered intravenously.
  • appropriate intravenous doses for an adult include about 60 mg/m2 to about 75 mg/m2 at about 21 -day intervals or about 25 mg/m2 to about 30 mg/m2 on each of 2 or 3 successive days repeated at about 3 week to about 4 week intervals or about 20 mg/m2 once a week.
  • the lowest dose should be used in elderly patients, when there is prior bone- marrow depression caused by prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs.
  • Nitrogen mustards are another suitable chemotherapeutic agent useful in the methods of the disclosure.
  • a nitrogen mustard may include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and/or ifosfamide, melphalan (L-sarcolysin), and chlorambucil.
  • HN2 mechlorethamine
  • cyclophosphamide and/or ifosfamide melphalan
  • L-sarcolysin L-sarcolysin
  • chlorambucil chlorambucil.
  • Cyclophosphamide CYTOXAN®
  • NEOSTAR® is available from Adria
  • Adria is another suitable chemotherapeutic agent.
  • Suitable oral doses for adults include, for example, about 1 mg/kg/day to about 5 mg/kg/day
  • intravenous doses include, for example, initially about 40 mg/kg to about 50 mg/kg in divided doses over a period of about 2 days to about 5 days or about 10 mg/kg to about 15 mg/kg about every 7 days to about 10 days or about 3 mg/kg to about 5 mg/kg twice a week or about 1.5 mg/kg/day to about 3 mg/kg/day.
  • the intravenous route is preferred.
  • the drug also sometimes is administered intramuscularly, by infiltration or into body cavities.
  • Additional suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluouracil; 5-FU) and floxuridine (fluorode- oxyuridine; FudR).
  • 5-FU may be administered to a subject in a dosage of anywhere between about 7.5 to about 1000 mg/m2. Further, 5-FU dosing schedules may be for a variety of time periods, for example up to six weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.
  • Gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co., “gemcitabine”), another suitable chemotherapeutic agent, is recommended for treatment of advanced and metastatic pancreatic cancer, and will therefore be useful in the present disclosure for these cancers as well.
  • the amount of the chemotherapeutic agent delivered to the patient may be variable.
  • the chemotherapeutic agent may be administered in an amount effective to cause arrest or regression of the cancer in a host, when the chemotherapy is administered with the construct.
  • the chemotherapeutic agent may be administered in an amount that is anywhere between 2 to 10,000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent.
  • the chemotherapeutic agent may be administered in an amount that is about 20 fold less, about 500 fold less or even about 5000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent.
  • chemotherapeutic s of the disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct, as well as for determination of effective dosages.
  • suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, etc.
  • In vitro testing may also be used to determine suitable combinations and dosages, as described in the examples.
  • Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies.
  • Tumor resection refers to physical removal of at least part of a tumor.
  • treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).
  • a cavity may be formed in the body.
  • Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy.
  • Such treatment may be repeated, for example, equal to any one of, at least any one of, at most any one of, about any one of, or between any two of every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
  • These treatments may be of varying dosages as well.
  • agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment.
  • additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population.
  • cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatments.
  • Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments.
  • Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy.
  • aspects of the present disclosure are directed to methods comprising treatment of a subject suffering from, or suspected of having, cancer.
  • the cancer is colon cancer, prostate cancer, lung cancer, melanoma, or breast cancer.
  • the cancer is lung cancer.
  • the lung cancer is non-small cell lung cancer.
  • the disclosed methods comprise treating a subject who currently has or has previously had resistance to immunotherapy. A subject may be identified as having resistance to immunotherapy using tests and diagnostic methods known in the art.
  • the disclosed methods comprise treating a subject suffering from a cancer with a combination of radiotherapy, a checkpoint inhibitor therapy, and a MER proto-oncogene tyrosine kinase (MerTK)-targeted nucleic acid.
  • MerTK proto-oncogene tyrosine kinase
  • cancers associated with resistance to immunotherapy are surprisingly and unexpectedly sensitive to treatment with a combination of a radiotherapy, a checkpoint inhibitor therapy, and a MerTK- targeted nucleic acid.
  • administering a radiotherapy, a checkpoint inhibitor therapy, and a MerTK-targeted nucleic acid was surprisingly found to decrease radiotherapy-induced immunosuppression of a systemic response to an immunotherapy treatment and to inhibit primary and secondary tumor growth in the subject.
  • a method for treating a subject suffering from cancer with a radiotherapy, a checkpoint inhibitor therapy, and a MerTK-targeted nucleic acid is disclosed.
  • a method for stimulating an immune-mediated response at a metastatic tumor site with a radiotherapy, a checkpoint inhibitor therapy, and a MerTK-targeted nucleic acid is disclosed.
  • the cancer is a cancer characterized as being resistant to immunotherapy (e.g., lung cancer).
  • the cancer is metastatic cancer.
  • the radiotherapy comprises external radiotherapy, internal radiotherapy, radioimmunotherapy, or intraoperative radiation therapy (IORT).
  • the external radiotherapy comprises three-dimensional conformal radiation therapy (3D-CRT), intensity modulated radiation therapy (IMRT), proton beam therapy, image-guided radiation therapy (IGRT), or stereotactic radiation therapy.
  • the internal radiotherapy comprises interstitial brachytherapy, intracavitary brachytherapy, or intraluminal radiation therapy.
  • the radiotherapy is administered to a primary tumor.
  • the checkpoint inhibitor therapy comprises a cytotoxic T- lymphocyte-associated protein 4 (CTLA4) inhibitor, a programmed cell death protein 1 (PD1) inhibitor, a programmed death-ligand 1 (PDL-1) inhibitor, a lymphocyte activation gene-3 (LAG3) inhibitor, or a T-cell immunoglobulin and mucin domain 3 (TIM-3) inhibitor.
  • CTLA4 cytotoxic T- lymphocyte-associated protein 4
  • PD1 inhibitor a programmed cell death protein 1
  • PDL-1 inhibitor is an anti-PDl antibody
  • the checkpoint inhibitor therapy comprises a PDL-1 inhibitor.
  • the PDL-1 inhibitor is an anti-PDL-1 antibody.
  • the checkpoint inhibitor therapy comprises a CTLA4inhibitor.
  • the CTLA4 inhibitor is an anti-CTLA4 antibody.
  • the MerTK-targeted nucleic acid is an antisense oligonucleotide, a single- stranded deoxyribonucleic acid molecule, a small interfering RNA, a small hairpin RNA, a viral vector, or a guide RNA. In some embodiments, the MerTK-targeted nucleic acid is an antisense oligonucleotide.
  • the MerTK-targeted nucleic acid comprises a sequence having equal to any one of, at least any one of, at most any one of, about any one of, or between any two of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
  • SEQ ID NO:1 SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO: 9.
  • the MerTK-targeted nucleic acid comprises a sequence having equal to any one of, at least any one of, at most any one of, about any one of, or between any two of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
  • the MerTK- targeted nucleic acid comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
  • the MerTK-targeted nucleic acid comprises SEQ ID NO:1. In some embodiments, the MerTK-targeted nucleic acid comprises SEQ ID NO:2. In some embodiments, the MerTK-targeted nucleic acid comprises SEQ ID NO:3. In some embodiments, the MerTK-targeted nucleic acid comprises SEQ ID NO:4. In some embodiments, the MerTK-targeted nucleic acid comprises SEQ ID NO:5. In some embodiments, the MerTK-targeted nucleic acid comprises SEQ ID NO:6.
  • the MerTK-targeted nucleic acid comprises SEQ ID NO:7. In some embodiments, the MerTK-targeted nucleic acid comprises SEQ ID NO: 8. In some embodiments, the MerTK-targeted nucleic acid comprises SEQ ID NO:9.
  • the disclosed methods comprise identifying one or more subjects as being candidates for treatment with a combination of a radiotherapy, a checkpoint inhibitor therapy, and a MerTK-targeted nucleic acid, based on current or former resistance to treatment with an immunotherapy.
  • a method comprising identifying a subject having cancer as being a candidate for treatment with a combination of a radiotherapy, a checkpoint inhibitor therapy, and a MerTK-targeted nucleic acid by determining that the subject currently has or previously had resistance to treatment with an immunotherapy.
  • the disclosed methods comprise determining an optimal cancer treatment for a subject with resistance to treatment with an immunotherapy.
  • a subject is given multiple types of cancer therapy, for example a cancer immunotherapy and a chemotherapy.
  • compositions comprising a MerTK-targeted nucleic acid.
  • the pharmaceutical compositions can further comprise one or more additional therapeutics, for example, a checkpoint inhibitor.
  • the therapy provided herein may comprise administration of a combination of therapeutic agents, such as a first cancer therapy (e.g., a radiotherapy), a second cancer therapy (e.g., an immunotherapy, for example, a checkpoint inhibitor therapy), and a third cancer therapy (e.g., a MerTK-targeted nucleic acid).
  • the therapies may be administered in any suitable manner known in the art.
  • the first, second, and third cancer treatment may be administered sequentially (at different times) or concurrently (at the same time or approximately the same time; also “simultaneously” or “substantially simultaneously”).
  • the first, second, and/or third cancer treatments are administered in a separate composition.
  • the first, second, and/or third cancer treatments are in the same composition.
  • the checkpoint inhibitor therapy and the MerTK-targeted nucleic acid are administered substantially simultaneously.
  • the radiotherapy, the checkpoint inhibitor therapy, and the MerTK-targeted antisense oligonucleotide are administered substantially simultaneously.
  • the checkpoint inhibitor therapy and MerTK-targeted nucleic acid are administered sequentially.
  • the checkpoint inhibitor therapy, and the MerTK-targeted nucleic acid are administered sequentially.
  • the MerTK-targeted nucleic acid is administered before administering the radiotherapy.
  • the MerTK- targeted nucleic acid is administered after administering the radiotherapy.
  • a first dose of the MerTK-targeted nucleic acid is administered before administering the radiotherapy and a second dose of the MerTK-targeted nucleic acid is administered after administering the radiotherapy.
  • Embodiments of the disclosure relate to compositions and methods comprising therapeutic compositions.
  • the different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions.
  • Various combinations of the agents may be employed.
  • the therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration.
  • the cancer therapy is administered intratumorally, intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
  • the appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.
  • the treatments may include various “unit doses.”
  • Unit dose is defined as containing a predetermined-quantity of the therapeutic composition.
  • the quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts.
  • a unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time.
  • a unit dose comprises a single administrable dose.
  • a single dose of the MerTK-targeted nucleic acid is administered. In some embodiments, multiple doses of the MerTK-targeted nucleic acid are administered. In some embodiments, the MerTK-targeted nucleic acid is administered at a dose of between 1 mg/kg and 5000 mg/kg. In some embodiments, the MerTK-targeted nucleic acid is administered at a dose of equal to any one of, at least any one of, at most any one of, about any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
  • a single dose of the checkpoint inhibitor is administered.
  • multiple doses of the checkpoint inhibitor are administered.
  • the checkpoint inhibitor is administered at a dose of between 1 mg/kg and 100 mg/kg. In some embodiments, the checkpoint inhibitor is administered at a dose of equal to any one of, at least any one of, at most any one of, about any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
  • the radiotherapy administered to the subject provides irradiation in a dose range of 0.5 Gy to 60 Gy. In some embodiments, the radiotherapy administered to the subject provides irradiation at a dose of equal to any one of, at least any one of, at most any one of, about any one of, or between any two of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,
  • the radiotherapy is administered in a single dose. In some embodiments, the radiotherapy is administered in a fractionated dose over a period of time of not more than one week. In some embodiments, the radiotherapy is delivered in a fractionated dose over a period of time of not more than three days.
  • the quantity to be administered depends on the treatment effect desired.
  • An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain embodiments, it is contemplated that doses in the range from 10 mg/kg to 200 mg/kg can affect the protective capability of these agents.
  • doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 pg/kg, mg/kg, pg/day, or mg/day or any range derivable therein.
  • doses can be administered at multiple times during a day, and/or on multiple days, weeks, or months.
  • the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 pM to 150 pM.
  • the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10 pM to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to 50 pM; or about 50 pM to 150 pM; or about 50
  • the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: equal to any one of, at least any one of, at most any one of, about any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
  • the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent.
  • the blood levels discussed herein may refer to the unmetabolized therapeutic agent.
  • Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.
  • dosage units of pg/kg or mg/kg of body weight can be converted and expressed in comparable concentration units of pg/ml or mM (blood levels), such as 4 pM to 100 pM. It is also understood that uptake is species and organ/tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.
  • kits containing compositions of the invention or compositions to implement methods of the invention.
  • kits can be used to evaluate one or more biomarkers.
  • a kit contains, contains equal to any one of, at least any one of, at most any one of, about any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more probes, primers or primer sets, synthetic molecules or inhibitors, or any value or range and combination derivable therein.
  • there are kits for evaluating biomarker activity in a cell are kits for evaluating biomarker activity in a cell.
  • Kits may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means.
  • Individual components may also be provided in a kit in concentrated amounts; in some embodiments, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as lx, 2x, 5x, lOx, or 20x or more.
  • Kits for using probes, synthetic nucleic acids, nonsynthetic nucleic acids, and/or inhibitors of the disclosure for prognostic or diagnostic applications are included as part of the disclosure.
  • any such molecules corresponding to any biomarker identified herein which includes nucleic acid primers/primer sets and probes that are identical to or complementary to all or part of a biomarker, which may include noncoding sequences of the biomarker, as well as coding sequences of the biomarker.
  • kits may include a sample that is a negative or positive control for methylation of one or more biomarkers.
  • any embodiment of the disclosure involving specific biomarker by name is contemplated also to cover embodiments involving biomarkers whose sequences are equal to any one of, at least any one of, at most any one of, about any one of, or between any two of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to the mature sequence of the specified nucleic acid.
  • the MerTK ASO used has the sequence AGTGATATAATGGTCA (SEQ ID NO:1), molecular weight of 5,451.5, and equilibrium constant of 173.44.
  • the inventors established a bilateral tumor model in 129Sv/Ev mice using a non-small cell lung cancer cell line, 344SQR, an aPDl -resistant tumor model in mice.
  • mice received several intraperitoneal injections of anti-PDl antibody and MerTK ASO, and in some cases, intratumoral injections of MerTK ASO.
  • the triple combination therapy (XRT+ anti-PDl antibody + MerTK ASO) achieved significant secondary tumor growth delay compared to other combination therapies.
  • mice were inoculated by subcutaneous injection of 0.5xl0 5 344SQR cells into the right hind legs for establishment of the primary tumor.
  • mice received another inoculation by subcutaneous injection of 0.5xl0 5 344SQR cells into the left hind legs for establishment of the secondary tumor.
  • Treatments with 10 mg/kg of anti-PDl antibody were performed on days 5, 8, 11, and 14 via intraperitoneal injection.
  • Treatments with 10 mg/kg of anti-PDl antibody (/nVzvoMAb antimouse PD-1 antibody (CD279), BioXcell) continued once a week from day 21.
  • XRT 36 Gy in three 12-Gy fractions
  • Treatments with 50 mg/kg MerTK ASO were performed on days 1, 2, 3, 4, 7, 8, 9, 10, 14, and 18 via intraperitoneal injection.
  • Treatments with 50 mg/kg MerTK ASO via intraperitoneal injection continued twice a week from day 14.
  • Intratumoral injection of 50 mg/kg MerTK ASO was performed on days 7 and 11.
  • the control group was inoculated with both primary and secondary tumors, but did not receive any treatment.
  • Table 1 below provides a summary of treatments received by eight different experimental groups.
  • Group 2 mice received an XRT dose of 36 Gy in three 12-Gy fractions.
  • Group 3 mice received 50 mg/kg MerTK ASO according to the dosing schedule of the experimental timeline of FIG. 1.
  • Group 4 mice received 10 mg/kg of anti-PDl antibody according to the dosing schedule of the experimental timeline of FIG. 1.
  • Group 5 mice received an XRT dose of 36 Gy in three 12- Gy fractions and 50 mg/kg MerTK ASO according to the dosing schedule of the experimental timeline of FIG. 1.
  • mice received an XRT dose of 36 Gy in three 12-Gy fractions and 10 mg/kg of anti-PDl antibody according to the dosing schedule of the experimental timeline of FIG. 1.
  • Group 7 mice received an XRT dose of 36 Gy in three 12-Gy fractions and 50 mg/kg MerTK ASO and 10 mg/kg of anti-PDl antibody according to the dosing schedule of the experimental timeline of FIG. 1.
  • Group 8 mice received an XRT dose of 36 Gy in three 12-Gy fractions, 50 mg/kg MerTK ASO injected intratumorally pre- and post-administration of all XRT dose fractions, and 10 mg/kg of anti-PDl antibody according to the dosing schedule of the experimental timeline of FIG. 1.
  • XRT+anti-PDl antibody+MertK IP resulted in better secondary tumor control than XRT+anti- PDl antibody+MertK IT .
  • XRT+anti-PDl antibody+MerTK IP group achieved the best survival rate in the mice.
  • DCs dendritic cells
  • CD45 + cells CD45 + cells in the primary tumor.
  • FIG. 12 shows that within the primary tumor, other than a slight increase in THI cells in mice treated with RCM (FIG. 12), there was no additional activation of any immune pathways gained from adding the MerTK ASO to any other therapeutic modality (FIG. 8A, FIG. 12).
  • MerTK inhibition with the ASO imparts no significant increase in immune activation at the primary tumor.
  • mice treated with the MerTK ASO were found to be upregulated within the secondary tumors of mice treated with the MerTK ASO, including, among others ROS generation, pattern recognition receptors signaling, complement and humoral immunity, adhesion and cell-cell interactions, inflammation, and NFKB signaling.
  • This relative immune gene upregulation was the most dramatic in the secondary tumors of mice treated with XRT+aCTLA4 (FIG. 14); compared to mice treated with XRT+aCTLA4 alone, mice treated with RCM exhibited a five-fold increase in the expression of several immune- related genes across multiple different pathways.
  • the immune activation gained from the addition of the MerTK ASO to mice treated with XRT+aPDl was more modest, but still substantial.
  • control of the primary tumor is principally mediated through the direct effects of the radiotherapy, whereas control of the secondary tumor is principally mediated by the immune system, which has been invigorated by the conversion of the radiation-damaged primary tumor into an in situ vaccine (Formenti and Demaria, 2012), and then further bolstered through the activity of MerTK inhibition.
  • Cell line. 344SQR an aPDl resistant lung cancer cell line
  • the cell line was cultured in complete medium (RPML1640 supplemented with 100 units/mL penicillin, 100 pg/mL streptomycin, and 10% heat-inactivated fetal bovine serum) and incubated at 37 °C in 5% CO 2 .
  • a MerTK-specific ASO with a sequence of AGTGATATAATGGTCA (SEQ ID NO:1) and an ASO control with a sequence of GGCTACTACGCCGTCA (SEQ ID NO: 10) were produced by lonis Pharmaceuticals, Inc.
  • Anti-mouse CTLA-4 (Catalog# BP0164) and anti-mouse PD-1 (Catalog# BE0146) were purchased from BioXCell.
  • Liberase Catalog #05401127001
  • DNAse Catalog #4716728001
  • Flow cytometry antibodies including CD45-PerCP Cy5.5 (Catalog# 103131), CD4-PE/Dazzle594 (Catalog# 100456), CD8-FITC (Catalog# 100706), Granzyme B (GrB)-Pacific Blue (Catalog# 515408), Grl-BV510 (Catalog# 108437), CDl lb-APC Fire750 (Catalog# 101262), F4/80- Alexa Fluor 700 (Catalog# 123130), CD38-PE-Cy7 (Catalog# 102718), CD206-PE (Catalog# 141706), and MertK-APC (Catalog# 151507) were ordered from BioEegend.
  • the dose was delivered with two opposing beams from the AP and PA positions using a 15 mm circular collimator.
  • the dosimetry and treatment planning were performed using the Advanced Treatment Planning software that is supplied by the vendor with the unit. All collimators were commissioned by Precision XRay Corporation at the time of installation. Routine output checks are performed using an ion chamber to ensure that the outputs have not changed and that the treatment plans continue to be accurate.
  • For the MerTK ASO administered intraperitoneally 50 mg/kg of MerTK ASO (MerTK) in PBS was injected to the mice on days 1, 2, 3, 4, 7, 8, 9, 10, 14, 18, 21, 25, 28, 32, 35 and 39. Ten mg/kg of aPDl was injected on days 5, 8, 11, 14, 21, 28, and 35 via intraperitoneal (IP) injection.
  • IP intraperitoneal
  • RNA-Based Therapeutics From Antisense Oligonucleotides to miRNAs. Cells 9.
  • Pan-TAM Tyrosine Kinase Inhibitor BMS-777607 Enhances Anti-PD-1 mAb Efficacy in a Murine Model of Triple-Negative Breast Cancer. Cancer Res 79, 2669-2683.

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Abstract

Des aspects de la présente invention concernent des procédés de traitement d'un sujet atteint d'un cancer. Certains aspects concernent le traitement avec une radiothérapie, une thérapie par inhibiteur de point de contrôle immunitaire, et un acide nucléique ciblant la tyrosine kinase proto-oncogène MER (MerTK), comprenant un oligonucléotide antisens ciblant MerTK. Dans certains cas, un sujet a été déterminé comme ayant ou ayant eu une résistance à un traitement de cancer précédent, tel qu'une immunothérapie. D'autres aspects concernent des procédés d'identification d'un sujet en tant que candidat pour un traitement par une combinaison d'une radiothérapie, d'une thérapie par inhibiteur de point de contrôle immunitaire et d'un acide nucléique ciblant MerTK.
PCT/US2021/050870 2020-09-18 2021-09-17 Traitement combiné pour le cancer Ceased WO2022061112A1 (fr)

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WO2025111378A1 (fr) * 2023-11-20 2025-05-30 Tautona Group Ip Holding Company, L.L.C. Formulations semi-solides de déféroxamine pour le traitement de brûlures de radiothérapie

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WO2024148175A1 (fr) * 2023-01-05 2024-07-11 Opus Genetics Inc. Thérapie génique pour une maladie oculaire
WO2025111378A1 (fr) * 2023-11-20 2025-05-30 Tautona Group Ip Holding Company, L.L.C. Formulations semi-solides de déféroxamine pour le traitement de brûlures de radiothérapie

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