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WO 2017/112943 Al 29 June 2017 (29.06.2017) W P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2017/112943 Al 29 June 2017 (29.06.2017) W P O P C T (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, C07K 14/705 (2006.01) A61K 31/7088 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, C12N 15/12 (2006.01) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KH, KN, (21) International Application Number: KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, PCT/US2016/068552 MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, (22) International Filing Date: NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, 23 December 2016 (23. 12.2016) RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, (25) Filing Language: English ZA, ZM, ZW. (26) Publication Language: English 4 Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 62/387,168 23 December 201 5 (23. 12.2015) US GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, 62/290,413 2 February 2016 (02.02.2016) US TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, (71) Applicant: MODERNATX, INC. [US/US]; 200 Techno DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, logy Square, Cambridge, MA 02139 (US). LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, (72) Inventors: FREDERICK, Joshua; 4 1 Garden Street., Apt. GW, KM, ML, MR, NE, SN, TD, TG). 5, Boston, MA 021 14 (US). BAI, Ailin; 63 Faxon Street, Newton, MA 02458 (US). Published: (74) Agents: KIM, Ji-Eun et al; Sterne, Kessler, Goldstein & — with international search report (Art. 21(3)) Fox P.L.L.C, 1100 New York Avenue, NW, Washington, — with sequence listing part of description (Rule 5.2(a)) DC 20005 (US). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (54) Title: METHODS OF USING OX40 LIGAND ENCODING POLYNUCLEOTIDES FIGURE 1 5' UTR ORF encoding OX40L Polypeptide 3' I Polv-A Tail f 5' Cap miR 2 binding site (57) Abstract: The disclosure relates to compositions and methods for the preparation, manufacture and therapeutic use of polynuc - © leotide molecules comprising an mRNA encoding an OX40L polypeptide. Also provided is a method for activating T cells or in- creasing the number of NK cells in a subject in need thereof. METHODS OF USING OX40 LIGAND ENCODING POLYNUCLEOTIDES BACKGROUND [0001] Cancer is a disease characterized by uncontrolled cell division and growth within the body. In the United States, roughly a third of all women and half of all men will experience cancer in their lifetime. Polypeptides are involved in every aspect of the disease including cancer cell biology (carcinogenesis, cell cycle suppression, DNA repair and angiogenesis), treatment (immunotherapy, hormone manipulation, enzymatic inhibition), and/or diagnosis and determination of cancer type (molecular markers for breast, prostate, colon and cervical cancer for example). With the host of undesired consequences brought about by standard treatments such as chemotherapy and radiotherapy used today, genetic therapy for the manipulation of disease-related peptides and their functions provides a more targeted approach to disease diagnosis, treatment and management. However, gene therapy poses multiple challenges including undesirable immune response and safety concern due to the incorporation of the gene at random locations within the genome. [0002] Various methods of treating cancer are under development. For example, dendritic cell (DC) vaccines have been studied as a possible anti-cancer therapy. However, DC vaccines require multiple steps of isolating DCs from a subject, ex vivo manipulation of DCs to prime the cells for tumor antigen presentation, and subsequent administration of the manipulated DCs back into the subject. Further, it is reported that the overall clinical response rates for DC vaccines remain low and the ability of DC vaccines to induce cancer regression remains low. See, e.g., Kalkinski et al, "Dendritic cell-based therapeutic cancer vaccines: what we have and what we need," Future Oncol. 5(3):379- 390 (2009). BRIEF SUMMARY [0003] The present disclosure relates to compositions and methods for activating an immune response in a subject. One aspect of the disclosure provides a method of activating T cells in a subject in need thereof comprising administering to the subject an effective amount of a polynucleotide comprising an mRNA encoding an OX40L polypeptide. In another aspect, the activated T cells reduce or decrease the size of a tumor or inhibit growth of a tumor in the subject. [0004] Another aspect of the disclosure provides a method of increasing the number of Natural Killer (NK) cells in a subject in need thereof comprising administering to the subject an effective amount of a polynucleotide comprising an mRNA encoding an OX40L polypeptide. In another aspect, the increased NK cells reduce or decrease the size of a tumor or inhibit growth of a tumor in the subject. [0005] In some embodiments, the disclosure provides a method for activating T cells and increasing the number of NK cells in a subject in need thereof comprising administering to the subject an effective amount of a polynucleotide comprising an mRNA encoding an OX40L polypeptide. [0006] In another embodiment, administering to the subject an effective amount of a polynucleotide comprising an mRNA encoding an OX40L polypeptide further induces IL-2 release. In another embodiment, administering to the subject an effective amount of a polynucleotide comprising an mRNA encoding an OX40L polypeptide further induces IL-4 release. In another embodiment, administering to the subject an effective amount of a polynucleotide comprising an mRNA encoding an OX40L polypeptide further induces IL-21 release. [0007] In some embodiments, administering to the subject an effective amount of a polynucleotide comprising an mRNA encoding an OX40L polypeptide induces T cell proliferation. In other embodiments, administering to the subject an effective amount of a polynucleotide comprising an mRNA encoding an OX40L polypeptide induces T cell infiltration in the tumor or increases the number of tumor infiltrating T cells. In certain embodiments, administering to the subject an effective amount of a polynucleotide comprising an mRNA encoding an OX40L polypeptide induces a memory T cell response. In some embodiments, the activated T cells comprise CD4+ T cells. In other embodiments the activated T cells comprise CD8+ T cells. In other embodiments, the activated T cells comprise both CD4+ T cell and CD8 T cells. In some embodiments, the number of NK cells is increased at least about two-fold, at least about three-fold, at least about four-fold, at least about five-fold, at least about six-fold, at least about seven-fold, at least about eight-fold, at least about nine-fold, or at least about ten-fold. [0008] In some embodiments of the disclosure, the polynucleotide comprises at least one chemically modified nucleoside as described herein. In one embodiment, the at least one chemically modified nucleoside comprises two or more combinations thereof. In another embodiment, the at least one chemically modified nucleoside is selected from the group consisting of pseudouridine (ψ), Nl-methylpseudouridine ( η ΐ ψ), 2-thiouridine (s2U), 4'- thiouridine, 5-methylcytosine, 2-thio-l -methyl- 1-deaza-pseudouri dine, 2-thio-l-methyl- pseudouridine, 2-thio-5-aza-uridine , 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio- 1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5- methyluridine, 5-methoxyuridine, 2'-0-methyl uridine, 1-methyl-pseudouridine (η ΐ ψ), 5- methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), a-thio-guanosine, a-thio-adenosine, 5-cyano uridine, 4'-thio uridine 7-deaza-adenine, 1-methyl-adenosine (mlA), 2-methyl- adenine (m2A), N6-methyl-adenosine (m6A), and 2,6-Diaminopurine, (I), 1-methyl- inosine (mil), wyosine (imG), methyl wyosine (mimG), 7-deaza-guanosine, 7-cyano-7- deaza-guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQl), 7-methyl- guanosine (m7G), 1-methyl-guanosine (mlG), 8-oxo-guanosine, 7-methyl-8-oxo- guanosine, and two or more combinations thereof. [0009] In another embodiment of the disclosure, the nucleosides in the mRNA are chemically modified by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% . In another aspect, the chemically modified nucleosides in the mRNA are selected from the group consisting of uridine, adenine, cytosine, guanine, and any combination thereof. [0010] In one embodiment, the uridine nucleosides in the mRNA are chemically modified by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%. In another embodiment, the adenine nucleosides in the mRNA are chemically modified by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%.
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