WO 2018/102397 Al 07 June 2018 (07.06.2018) W !P O PCT
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(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 2018/102397 Al 07 June 2018 (07.06.2018) W !P O PCT (51) International Patent Classification: John; 64 Gray Street, Arlington, Massachusetts 02476 A61K 9/133 (2006.01) A61K 9/10 (2006.01) (US). MUTAMBA, James Tendai; 70 Longfellow Road, A61K 9/127 {2006.01) Newton, Massachusetts 02462 (US). SHYAM, Rishab R.; 995 Massachusetts Avenue, Arlington, Massachusetts (21) International Application Number: 02476 (US). PCT/US2017/063681 (74) Agent: RED), Andrea L.C. et al; One International (22) International Filing Date: Place, 40th Floor, 100 Oliver Street, Boston, Massachusetts 29 November 201 7 (29. 11.201 7) 021 10-2605 (US). (25) Filing Language: English (81) Designated States (unless otherwise indicated, for every (26) Publication Langi English kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, (30) Priority Data: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, 62/427,53 1 29 November 2016 (29. 11.2016) US DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, 62/559,921 18 September 2017 (18.09.2017) US HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, 62/559,967 18 September 2017 (18.09.2017) US KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, (71) Applicant: PURETECH HEALTH LLC [US/US]; 501 MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, Boylston Street, Suite 6102, Boston, Massachusetts 021 16 OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (US). SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (72) Inventors: BOLEN, Joseph; 303 3rd Street, Cambridge, Massachusetts 02142 (US). BONNER, Daniel Kenneth; (84) Designated States (unless otherwise indicated, for every 107 Edgemont Road, Braintree, Massachusetts 02144 (US). kind of regional protection available): ARIPO (BW, GH, FERREIRA, Lisa V.; 1 Highwood Drive, Franklin, Mass GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, achusetts 02038 (US). KRUMOVA, Katerina; 2 Orchard UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, Terrace, Swampscott, Massachusetts 01907 (US). JANTZ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (54) Title: EXOSOMES FOR DELIVERY OF THERAPEUTIC AGENTS (57) Abstract: The present invention provides exosomes as drug de FIG. livery vehicles, compositions comprising a therapeutic agent encapsu lated within such exosomes, methods of producing such exosomes and compositions thereof, as well as methods of delivering such exosomes and compositions to a specific patient tissue or organ. The present in vention also provides methods of treating a disease, disorder, or con dition such as cancer, an inflammatory disease, an infectious disease, an allergic disease, or an autoimmune disease, comprising administer ing to a patient in need thereof a provided therapeutic-loaded exosome or a pharmaceutical composition thereof. siRNA-DY677 and exosome interaction - outside Ch-siRNA-DY677 and exosome interaction - surface + inside [Continued on nextpage] WO 2018/102397 Al llll II II 11III II I 11II I III 11 II III II I II EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, 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, GW, KM, ML, MR, NE, SN, TD, TG). Published: — with international search report (Art. 21(3)) — before the expiration of the time limit for amending the claims and to be republished in the event of receipt of amendments (Rule 48.2(h)) EXOSOMES FOR DELIVERY OF THERAPEUTIC AGENTS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application nos. 62/427,531, filed November 29, 2016; 62/559,921, filed September 18, 2017; and 62/559,967, filed September 18, 2017; the contents of all of which are incorporated herein in their entireties by reference. TECHNICAL FIELD [0002] The present invention relates, in part, to microvesicles, e.g. exosomes, capable of loading (e.g., encapsulating) therapeutic agents, for example biologies such as proteins, nucleic acids, or other agents, and, in some embodiments, improving their stability or other properties and/or delivering them to a tissue or organ in a patient. The present invention also relates to compositions and methods of using such microvesicles. BACKGROUND OF THE INVENTION [0003] Recent years have seen tremendous development of biologies and related therapeutic agents to treat, diagnose, and monitor disease. However, the challenge of generating suitable vehicles to package, stabilize and deliver payloads to sites of interest remains unaddressed. Many therapeutics suffer from degradation due to their inherent instability and active clearance mechanisms in vivo. Poor in vivo stability is particularly problematic when delivering these payloads orally. The harsh conditions of the digestive tract, including acidic conditions in the stomach, peristaltic motions coupled with the action of proteases, lipases, amylases, and nucleases that break down ingested components in the gastrointestinal tract, make it particularly challenging to deliver many biologies orally. The scale of this challenge is evidenced by the number of biologies limited to delivery via non-oral means, including IV, transdermal, and sub cutaneous administration. A general oral delivery vehicle for biologies and related therapeutic agents would profoundly impact healthcare. [0004] Recent efforts have focused on the packaging of biologies into polymer-based, liposomal, or biodegradable and erodible matrices that result in biologic-encapsulated nanoparticles. Despite their advantageous encapsulation properties, such nanoparticles have not achieved widespread use due to toxicity or poor release properties. Additionally, current nanoparticle synthesis techniques are limited in their ability to scale for manufacturing purposes. The development of an effective, non-toxic, and scalable delivery platform thus remains an unmet need. [0005] Exosomes (a class of microvesicles), which until fairly recently were thought of as cellular garbage containers, have emerged as entities known to play a key role in the communication of biological messages and the maintenance of physiological homeostasis. This means of biological communication seems to be conserved across many organisms, and includes the transport of various biomolecules including nucleic acids, proteins, and small molecules. [0006] Milk, which is orally ingested and known to contain a variety of miRNAs important for immune development, protects and delivers these miRNAs in exosomes. Milk exosomes therefore represent a gastrointestinally-privileged (Gl-privileged), evolutionarily conserved means of communicating important messages from mother to baby while maintaining the integrity of these complex biomolecules. Indeed, when compared with other types of exosomes, milk exosomes have been observed to have a favorable stability profile at acidic pH and other high-stress or degradative conditions See, e.g., IntJ Biol Sci. 2012;8(1): 118-23. Epub 201 1 Nov 29). Additionally, bovine miRNA levels in circulation have been observed to increase in a dose- dependent manner after consuming varying quantities of milk (See, e.g., PLoS One 2015; 10(3): e0121 123). [0007] Collectively, the available data suggest that humans have the ability to absorb intact nucleic acid contents of milk. Since milk exosomes are known to encapsulate miRNA species {See, e.g., J Nutr. 2014 Oct; 144(10): 1495-500) appropriate milk exosomes would enable oral delivery of a variety of therapeutic agents. Concordant with this hypothesis, poorly orally available small molecules have been packaged in milk exosomes and delivered orally in rodent models {See, e.g., Cancer Lett 2016 Feb 1;371(1):48-61). [0008] The present invention harnesses milk-derived exosomes to meet the urgent need for suitable delivery vehicles for therapeutics that were previously not orally administrable or suffered from other delivery challenges such as poor bioavailability, storage instability, metabolism, off-target toxicity, or decomposition in vivo. SUMMARY OF THE INVENTION [0009] In one aspect, the present invention provides microvesicles, such as milk-derived exosomes, as vehicles for therapeutic agents such as DNA, RNA, iRNA and antisense oligonucleotides and analogs of nucleic acids, antibodies, hormones, and other peptides and proteins. In some embodiments, the therapeutic agent is conjugated to a hydrophobic group such as a sterol, steroid, or lipid. In some embodiments, the hydrophobic group facilitates loading of the therapeutic agent into the exosome and/or delivery of the therapeutic agent to a target tissue or organ. The microvesicles may be loaded with a therapeutic agent through a variety of different methods disclosed herein. In one aspect, the present invention provides a therapeutic agent-loaded exosome ("therapeutic-loaded exosome") and pharmaceutical compositions comprising the same. In certain embodiments, provided exosomes are useful for delivery of an effective amount of a therapeutic agent to a patient in need thereof for the diagnosis, prevention, treatment, prognosis, or monitoring of disease. Such therapeutic-loaded exosomes and methods of using the same are described in detail, herein. BRIEF DESCRIPTION OF THE FIGURES [0010] FIG. 1 shows a distribution curve of milk exosome diameters for exosomes isolated from colostrum and raw milk. [0011] FIG. 2 shows a Cryo-TEM image of a milk exosome. [0012] FIG. 3 shows results demonstrating that isolated milk exosomes contain CD81, a classical exosome tetraspanin. [0013] FIG. 4 shows the results of a 14-day stability study. Protein concentration was measured each day for a sample stored at 4 °C (upper graph). Protein concentrations were also measured at day 14 for samples stored at room temperature, 4 °C, -20 °C, and -80 °C, respectively (lower graph).