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Ep 3039174 B1 (19) TZZ¥Z¥__T (11) EP 3 039 174 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12Q 1/6886 (2018.01) C12Q 1/6883 (2018.01) 16.10.2019 Bulletin 2019/42 (86) International application number: (21) Application number: 14840036.9 PCT/US2014/053306 (22) Date of filing: 28.08.2014 (87) International publication number: WO 2015/031694 (05.03.2015 Gazette 2015/09) (54) OLIGONUCLEOTIDE PROBES AND USES THEREOF OLIGONUKLEOTIDSONDEN UND VERWENDUNGEN DAVON SONDES OLIGONUCLÉOTIDIQUES ET LEURS UTILISATIONS (84) Designated Contracting States: (74) Representative: Patent Boutique LLP AL AT BE BG CH CY CZ DE DK EE ES FI FR GB 10A Printing House Yard GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Hackney Road PL PT RO RS SE SI SK SM TR London E2 7PR (GB) (30) Priority: 28.08.2013 US 201361871107 P (56) References cited: 06.09.2013 US 201361874621 P WO-A1-2011/066589 WO-A2-2010/056337 06.11.2013 US 201361900975 P WO-A2-2011/088226 WO-A2-2013/022995 05.12.2013 US 201361912471 P 06.01.2014 US 201461924192 P • TERESA JANAS ET AL: "The selection of 06.03.2014 US 201461949216 P aptamers specific for membrane molecular 03.04.2014 US 201461974949 P targets", CELLULAR & MOLECULAR BIOLOGY 07.05.2014 US 201461990085 P LETTERS, vol. 16, no. 1, 1 March 2011 16.05.2014 US 201461994704 P (2011-03-01), pages 25-39, XP055001923, ISSN: 14.07.2014 US 201462024436 P 1425-8153, DOI: 10.2478/s11658-010-0023-3 • HENNING ULRICH ET AL: "Disease-specific (43) Date of publication of application: biomarkerdiscovery by aptamers", CYTOMETRY 06.07.2016 Bulletin 2016/27 PART A, vol. 75A, no. 9, 1 September 2009 (2009-09-01), pages727-733, XP055032998, ISSN: (73) Proprietor: Caris Science, Inc. 1552-4922, DOI: 10.1002/cyto.a.20766 Irving, TX 75039 (US) • MAO YE ET AL: "Generating Aptamers by Cell-SELEX for Applications in Molecular (72) Inventors: Medicine", INTERNATIONAL JOURNAL OF • SPETZLER, David MOLECULARSCIENCES, vol. 13, no. 12, 12 March Paradise Valley, AZ 85253 (US) 2012 (2012-03-12), pages 3341-3353, • DOMENYUK, Valeriy XP055241055, DOI: 10.3390/ijms13033341 Phoenix, AZ 85042 (US) • SEFAH ET AL.: ’DEVELOPMENT OF DNA • XIAO, Nianqing APTAMERS USING CELL -SELEX’ NAT PROTOC Rockville, MD 20850 (US) vol. 5, no. 6, 2010, pages 1169 - 1185, • STARK, Adam XP055257422 Phoenix, AZ 85040 (US) • TROY ET AL.: ’Understanding barriers to Borrelia • ZHONG, Zhenyu burgdorferi dissemination during infection using Peoria, AZ 85383 (US) massively parallel sequencing.’ INFECT IMMUN vol. 81, no. 7, July 2013, pages 2347 - 2357, XP055257424 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 3 039 174 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 3 039 174 B1 Remarks: The complete document including Reference Tables and the Sequence Listing can be downloaded from the EPO website 2 EP 3 039 174 B1 Description BACKGROUND 5 [0001] The disclosure relates generally to the field of aptamers capable of binding to microvesicle surface antigens, which are useful as therapeutics in and diagnostics of cancer and/or other diseases or disorders in which microvesicles implicated. The disclosure further relates to materials and methods for the administration of aptamers capable of binding to microvesicles. The microvesicles may be derived from cells indicative of cancer, e.g., a breast cancer. [0002] Aptamers are multi-meric nucleic acid molecules having specific binding affinity to molecules, which may be 10 through interactions other than classic Watson-Crick base pairing. The terms aptamer, oligonucleotide, polynucleotide, or the like may be used interchangeably herein. [0003] Aptamers, like peptides generated by phage display or monoclonal antibodies ("mAbs"), are capable of spe- cifically binding to selected targets and modulating the target’s activity, e.g., through binding aptamers may block their target’s ability to function. Created by an in vitro selection process from pools of random sequence oligonucleotides, 15 aptamers have been generated for over 100 proteins including growth factors, transcription factors, enzymes, immu- noglobulins, and receptors. A typical aptamer is 10-15 kDa in size (30-45 nucleotides), binds its target with sub-nanomolar affinity, and discriminates against closely related targets (e.g., aptamers will typically not bind other proteins from the same gene family). A series of structural studies have shown that aptamers are capable of using the same types of binding interactions (e.g., hydrogen bonding, electrostatic complementarity, hydrophobic contacts, steric exclusion) that 20 drive affinity and specificity in antibody-antigen complexes. [0004] Aptamers have a number of desirable characteristics for use as therapeutics and diagnostics including high specificity and affinity, biological efficacy, and excellent pharmacokinetic properties. In addition, they offer specific com- petitive advantages over antibodies and other protein biologies, for example: [0005] Speed and control. Aptamers are produced by an entirely in vitro process, allowing for the rapid generation of 25 initial leads, including therapeutic leads. In vitro selection allows the specificity and affinity of the aptamer to be tightly controlled and allows the generation of leads, including leads against both toxic and non-immunogenic targets. [0006] Toxicity and Immunogenicity. Aptamers as a class have demonstrated little or no toxicity or immunogenicity. In chronic dosing of rats or woodchucks with high levels of aptamer (10 mg/kg daily for 90 days), no toxicity is observed by any clinical, cellular, or biochemical measure. Whereas the efficacy of many monoclonal antibodies can be severely 30 limited by immune response to antibodies themselves, it is extremely difficult to elicit antibodies to aptamers most likely because aptamers cannot be presented by T-cells via the MHC and the immune response is generally trained not to recognize nucleic acid fragments. [0007] Administration. Whereas most currently approved antibody therapeutics are administered by intravenous infu- sion (typically over 2-4 hours), aptamers can be administered by subcutaneous injection (aptamer bioavailability via 35 subcutaneous administration is >80% in monkey studies (Tucker et al., J. Chromatography B. 732: 203-212, 1999)). This difference is primarily due to the comparatively low solubility and thus large volumes necessary for most therapeutic mAbs. With good solubility (>150 mg/mL) and comparatively low molecular weight (aptamer: 10-50 kDa; antibody: 150 kDa), a weekly dose of aptamer may be delivered by injection in a volume of less than 0.5 mL. In addition, the small size of aptamers allows them to penetrate into areas of conformational constrictions that do not allow for antibodies or 40 antibody fragments to penetrate, presenting yet another advantage of aptamer-based therapeutics or prophylaxis. [0008] Scalability and cost. Aptamers are chemically synthesized and are readily scaled as needed to meet production demand for diagnostic or therapeutic applications. Whereas difficulties in scaling production are currently limiting the availability of some biologics and the capital cost of a large-scale protein production plant is enormous, a single large- scale oligonucleotide synthesizer can produce upwards of 100 kg/year and requires a relatively modest initial investment. 45 The current cost of goods for aptamer synthesis at the kilogram scale is estimated at $100/g, comparable to that for highly optimized antibodies. [0009] Stability. Aptamers are chemically robust. They are intrinsically adapted to regain activity following exposure to factors such as heat and denaturants and can be stored for extended periods (>1 yr) at room temperature as lyophilized powders. 50 [0010] WO 2010/056337 A2 (CARIS MPI) discloses a set of aptamers with the capacity of characterizing a disease in a sample of microvesicles derived from a patient’s sample (e.g. paragraphs [0731] - [0769]). SUMMARY OF THE INVENTION 55 [0011] Compositions and methods provide aptamers that bind biomarkers of interest such as microvesicle surface antigens or functional fragments of microvesicle surface antigens. Aptamers may be used in diagnostic, prognostic or theranostic processes to screen a biological sample for the presence or levels of microvesicle surface antigens determined to provide a diagnostic readout. The diagnosis may be related to a cancer, e.g., a breast cancer. Aptamers may be 3 EP 3 039 174 B1 chemically modified or composed in a pharmaceutical composition for therapeutic applications. [0012] In an aspect, the disclosure provides an oligonucleotide at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100 percent homologous to SEQ ID NO. 10558. In a related aspect, the disclosure provides a plurality of oligonucleotides comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 45, 50, 5 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 106, 107, 108, 109,
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