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WO 2014/100434 Al 26 June 2014 (26.06.2014) W P O P C T

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WO 2014/100434 Al 26 June 2014 (26.06.2014) 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 2014/100434 Al 26 June 2014 (26.06.2014) W P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12Q 1/68 (2006.0 1) G01N 33/566 (2006.0 1) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (21) International Application Number: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, PCT/US20 13/0766 11 DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (22) International Filing Date: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, 19 December 2013 (19. 12.2013) KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (25) Filing Language: English OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (26) Publication Language: English 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, (30) Priority Data: ZW. 61/739,558 19 December 2012 (19. 12.2012) US (84) Designated States (unless otherwise indicated, for every (71) Applicant: CARIS SCIENCE, INC. [US/US]; 6655 N. kind of regional protection available): ARIPO (BW, GH, MacArthur Boulevard, Irving, TX 75039 (US). GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (72) Inventors: SPETZLER, David; 6045 N. 40th St., Para TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, dise Valley, AZ 85253 (US). DOMENYUK, Valeriy; EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, 1249 East Spence Ave, Apt. 218, Tempe, AZ 85281 (US). MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, HORNUNG, Tassilo; 1249 East Spence Ave, Apt. 218, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, Tampe, AZ 85271 (US). MAYER, Giinter; Limes Insti KM, ML, MR, NE, SN, TD, TG). tute, University of Bonn, Domagk-Str. 1, 53 121 Bonn (DE). FAMULOK, Michael; Auerhofstrasse 4, 53 175 Published: Bonn (DE). — with international search report (Art. 21(3)) (74) Agent: AKHAVAN, Ramin; Caris Science, Inc., 6655 N. — with sequence listing part of description (Rule 5.2(a)) MacArthur Blvd., Irving, TX 75039 (US). o o- (54) Title: COMPOSITIONS AND METHODS FOR APTAMER SCREENING (57) Abstract: Methods are provided for selecting aptamers that are specific to a target of interest from amongst a library of potential aptamer sequences. Aptamers disclosed can be used to detect and/or characterize biological entities of interest, e.g. microvesicles ¾ and/or surface antigens. Further disclosed are biomarkers that can be used for diagnosing different disorders including different types S of cancer. COMPOSITIONS AND METHODS FOR APTAMER SCREENING CROSS REFERENCE [0001] This application claims the benefit of U.S. Provisional Patent Application Nos. 61/739,558, filed December 19, 2012; which application is incorporated herein by reference in its entirety. SEQUENCE LISTING SUBMITTED VIA EFS-WEB [0002] The entire content of the following electronic submission of the sequence listing via the USPTO EFS- WEB server, as authorized and set forth in MPEP §1730 II.B.2(a), is incorporated herein by reference in its entirety for all purposes. The sequence listing is within the electronically filed text file that is identified as follows: [0003] File Name : 37901815601SeqList.txt [0004] Date of Creation: December 19, 2013 [0005] Size (bytes) : 4,327 bytes BACKGROUND OF THE INVENTION [0006] The invention relates generally to the field of aptamers capable of binding to biomarkers such as microvesicle surface antigens, which are useful as therapeutics in and diagnostics of cancer and/or other diseases or disorders in which microvesicles implicated. The invention 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. [0007] Aptamers are nucleic acid molecules having specific binding affinity to molecules through interactions other than classic Watson-Crick base pairing. [0008] Aptamers, like peptides generated by phage display or monoclonal antibodies ("mAbs"), are capable of specifically 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, aptamers have been generated for over 100 proteins including growth factors, transcription factors, enzymes, immunoglobulins, 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 drive affinity and specificity in antibody-antigen complexes. [0009] 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 competitive advantages over antibodies and other protein biologies, for example: [0010] Speed and control. Aptamers are produced by an entirely in vitro process, allowing for the rapid generation of 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. [0011] 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 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. [0012] Administration. Whereas most currently approved antibody therapeutics are administered by intravenous infusion (typically over 2-4 hours), aptamers can be administered by subcutaneous injection (aptamer bioavailability via 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 antibody fragments to penetrate, presenting yet another advantage of aptamer-based therapeutics or prophylaxis. [0013] 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 biologies 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. The current cost of goods for aptamer synthesis at the kilogram scale is estimated at SlOO/g, comparable to that for highly optimized antibodies. [0014] 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. INCORPORATION BY REFERENCE [0015] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. SUMMARY OF THE INVENTION [0016] Compositions and methods of the invention provide aptamers that are useful in assays to detect biomarkers. One aspect of the invention provides methods of selecting aptamers of interest that specifically bind to one or more antigens in a biological sample of interest. In some embodiments of the invention, an aptamer or a plurality of aptamer molecules are identified, which are configured to bind to or associate with a plurality of target molecules present in one or more biological samples. The biological sample may comprise a population or subpopulation of microvesicles as further described herein. [0017] In an aspect, the invention provides a method of identifying one or more aptamer comprising: (a) providing a pool of candidate aptamers; (b) contacting a first one or more biological sample with the pool provided in step (a), wherein the first one or more biological sample comprises a heterogeneous microvesicle
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