Tunable cytotoxic aptamer–drug conjugates for thetreatment of prostate cancerBethany Powell Graya, Linsley Kellya,b, Douglas P. Ahrensc, Ashley P. Barryc, Christina Kratschmerb, Matthew Levyb,1,and Bruce A. Sullengera,2

aDepartment of Surgery, Duke University Medical Center, Durham, NC 27710; bDepartment of Biochemistry, Albert Einstein College of Medicine, Bronx, NY10461; and cResearch and Development Division, b3 bio, inc., Durham, NC 27709

Edited by Ronald R. Breaker, Yale University, New Haven, CT, and approved March 26, 2018 (received for review October 9, 2017)

Therapies that can eliminate both local and metastatic prostatetumor lesions while sparing normal organ tissue are desperatelyneeded. With the goal of developing an improved drug-targetingstrategy, we turned to a new class of targeted anticancertherapeutics: aptamers conjugated to highly toxic chemothera-peutics. Cell selection for aptamers with prostate cancer specificityyielded the E3 aptamer, which internalizes into prostate cancercells without targeting normal prostate cells. Chemical conjugationof E3 to the drugs monomethyl auristatin E (MMAE) and mono-methyl auristatin F (MMAF) yields a potent cytotoxic agent thatefficiently kills prostate cancer cells in vitro but does not affectnormal prostate epithelial cells. Importantly, the E3 aptamertargets tumors in vivo and treatment with the MMAF-E3 conjugatesignificantly inhibits prostate cancer growth in mice, demonstrat-ing the in vivo utility of aptamer–drug conjugates. Additionally,we report the use of antidotes to block E3 aptamer–drug conju-gate cytotoxicity, providing a safety switch in the unexpectedevent of normal cell killing in vivo.

aptamer | aptamer–drug conjugate | prostate cancer | drug targeting |antidote control

Prostate cancer is both the most commonly diagnosed malig-nancy and the second most common cause of cancer-related

mortality for men in the United States (1). Therapies thateliminate both local and metastatic prostate tumor legions whilesparing normal organ tissue are desperately needed. Recentexciting oncology advances have come from an old idea: anti-body–drug conjugates (ADCs). While the idea was first tested30 y ago (reviewed in ref. 2), it is only the development of new,highly toxic chemotherapy drugs coupled with improved anti-body–drug linker chemistry that has led to Food and Drug Ad-ministration approval of ADCs such a brentuximab vedotin(anti-CD30–MMAE) and ado-trastuzumab emtansine (anti-HER2–DM1). Several ADCs are currently in clinical trials for prostatecancer, including both a prostate-specific membrane antigen (PSMA)antibody (3–5) and an SCL44A4 antibody (6, 7) conjugated tothe highly toxic drug monomethyl auristatin E (MMAE). In allthese cases, the molecular target of the antibody is a known receptorup-regulated on the target cancer cell.Unfortunately almost all of the antigens that are recognized by

antibodies and ADCs are not exclusively expressed on cancercells, but also expressed at lower levels in normal tissue (8).Therefore, not surprisingly, toxicities have been observed withantibody-targeted therapies, resulting, at least in part, from an-tibody accumulation in normal tissue (9–12). For example,HER2-targeting antibodies are associated with significant car-diovascular toxicities due to HER2 expression in cardiac tissue(11), and epidermal growth factor receptor (EGFR)-targetedantibodies induce significant cutaneous toxicities (9, 10) due tocutaneous expression of EGFR. Therefore, to reduce patientmorbidity and improve the therapeutic window of targeted cy-totoxic agents, improved drug-targeting strategies are neededthat further focus cytotoxic agents toward cancer cells andminimize drug delivery to normal cells expressing even low levelsof target receptor.

While antibodies require humanization and are difficult tochemically modify with precision, aptamers, small RNA or DNAligands, have emerged as targeting agents with antibody-like af-finity (picomolar to nanomolar range) (reviewed in refs. 13–15)and the added benefit of ease of chemical synthesis and modifi-cation. Importantly, aptamers, unless formulated with poly-ethyleneglycol (PEG) (15), have thus far proven to be nontoxic,even when delivered to animals and humans at high doses (16–19).Traditionally aptamers are generated in the laboratory through aprocess known as Systematic Evolution of Ligands by EXponentialenrichment (SELEX), whereby a random oligonucleotide libraryis repetitively bound with a protein target of interest (20, 21). Inrecent years, a Cell-Internalization SELEX technique, involving acell target instead of a protein target, has been used to identifyoligonucleotides that internalize into different cell types (reviewedin ref. 22). Using two independent Cell-Internalization selectionsagainst a variety of different prostate cancer cell lines, we identi-fied an RNA aptamer, termed E3, specific for internalization intoprostate cancer, but not normal prostate cells.As a recent report suggests that aptamer–drug conjugates that

utilize highly toxic drugs can kill cells in vitro (23), we sought todevelop and evaluate the in vivo activity of E3–drug conjugates todetermine if this approach has clinical potential. We chemicallyconjugated E3 to the auristatin drugs MMAE and monomethylaruistatin F (MMAF) that have been successful in tumor targetingantibody conjugates. Both auristatin drugs are potent microtubuleinhibitors that are too toxic to be used in an unconjugated state(24, 25). The MMAE-E3 and MMAF-E3 aptamer conjugates

Significance

As prostate cancer is the second leading cause of cancer-relateddeath among men in the United States, an unmet medical needexists for therapies that eliminate prostate tumors while pre-venting toxicity in normal tissue. Recently, such tumor-targetingtherapies have gained Food and Drug Administration approval inthe form of antibody drug conjugates. However, a need exists fornew tumor-targeting therapies that are easier to manipulate andsynthesize. By selecting for RNA ligands that internalize intoprostate cancer cells but not normal prostate cells, we developedanother class of targeted agents. We demonstrate that the E3RNA selectively internalizes into prostate cancer cells and thatE3 highly toxic drug conjugates are potent anti-tumor agents.

Author contributions: B.P.G., M.L., and B.A.S. designed research; B.P.G., L.K., D.P.A., A.P.B.,and C.K. performed research; B.P.G., L.K., D.P.A., A.P.B., and C.K. analyzed data; and B.P.G.wrote the paper.

Conflict of interest statement: b3 bio, inc. (D.P.A. and A.P.B.) and Duke University (B.P.G.and B.A.S.) have submitted patent applications.

This article is a PNAS Direct Submission.

Published under the PNAS license.1Present address: Vitrisa Therapeutics, Durham, NC 27701.2To whom correspondence should be addressed. Email: bruce.sullenger@duke.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1717705115/-/DCSupplemental.

Published online April 16, 2018.

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efficiently killed prostate cancer cells in culture, but not normalprostate cells, with IC50s in the nanomolar range.We additionally aimed to exploit the unique ability of com-

plementary antidote oligonucleotides to exquisitely and rapidlycontrol aptamer activity (26, 27) so as to prevent unwantedaptamer targeting to normal cells if needed, as we have observedthat these antidotes effectively turn off aptamer targeting andfunction in humans within minutes of injection (15, 28, 29).Herein, we demonstrate that E3 targeting and internalizationcan also be rapidly turned off via antidote addition. Treatmentwith antidote also neutralizes both the MMAE-E3 and MMAFconjugates and prevents cell death in vitro, providing a safetyswitch in the rare case that our E3 conjugates were to experiencethe same type of unwanted accumulation in normal tissue, as hasbeen seen with antibody conjugates.Most importantly, both the E3 aptamer and MMAF-E3 drug

conjugate target and localize to prostate tumors in vivo, withMMAF-E3 significantly inhibiting tumor growth without en-gendering obvious toxicities. Thus, these animal studies indicatethat conjugation of internalizing aptamers to highly toxic che-motherapeutic agents yields a class of targeted anticancer ther-apeutics, and the MMAF-E3 conjugate represents a targetedapproach to treat patients with prostate cancer.

ResultsSelection of an RNA Aptamer That Internalizes into Prostate CancerCells. In an unbiased approach to identify RNA aptamers thatinternalize into prostate cancer cells at various stages of malig-nancy, we performed two Cell-Internalization SELEX using a 2′fluoro pyrimidine modified (30) RNA library comprising a mod-ified, nuclease-resistant RNA pool of ∼1014 different RNAs. Twodifferent selection strategies were employed. In the first, positiveselections targeting PC-3 prostate cancer cells were combined witha strong negative selection against normal prostate epithelial cells(PrEC) to deplete RNA molecules capable of internalizing intononcancerous cells. In the second, the cell target for the positiveselection was varied and included toggling between the LNCaP,PC-3, DU 145, and 22Rv1 cell lines (Fig. 1A and SI Appendix,Tables S1 and S2). These four prostate cancer cell lines representdifferent stages of prostate cancer progression, ranging from highsensitivity and dependence upon androgen hormones (LNCaPcells), to low androgen sensitivity (22Rv1 cells), to androgen in-dependence (PC-3 cells), and finally to a brain metastasis de-rivative (DU 145). Additionally, to exclude PSMA as a target, wechose to select against both PSMA-positive (LNCaP and 22Rv1)and PSMA-negative (PC-3 and DU 145) cell lines. Here again,positive rounds of selection targeting these cells were combinedwith a strong negative selection against PrECs. For both selec-tions, after incubating cells with the library of RNA variants andwashing away nonbinding aptamers, cell-surface-bound RNA wasdegraded via treatment with a harsh RNase mixture that degrades2′fluoro modified RNA (31), leaving only RNAs that had in-ternalized into cells. Subsequent reverse transcription, PCR am-plification, and transcription of the internalized RNA completed around of internalization selection.To assess selection progress, RNA pools from different se-

lection rounds were transcribed with a 3′ 22-nt tail that annealedto an Alexa Fluor 647-labeled reverse primer. Subsequent flowcytometry analysis clearly demonstrated an increase of in-ternalizing aptamers as the selection progressed through addi-tional rounds, as shown for the PC-3 cell selection (Fig. 1B).After nine rounds of selection against PC-3 cells, 12 uniqueRNAs were evaluated; two of them, the E3 and A3 aptamers,were found to internalize (SI Appendix, Table S3). Interestingly,after 14 rounds of toggling between LNCaP, PC-3, DU 145, and22Rv1 cells, the E3 and A3 aptamers also emerged as well as oneadditional internalizing aptamer (D11, SI Appendix, Table S3).Unexpectedly, the E3, A3, and D11 aptamers all competed forcellular binding and internalization, with the E3 aptamer con-sistently reaching the highest levels of cellular internalization.Therefore, the E3 aptamer was chosen for further studies.

The 83-nt-long E3 aptamer folds into two predicted secondarystructures (SI Appendix, Fig. S1). Truncation from the 5′ and 3′ends of the RNA results in a 36-nt minimized E3 structure that ispredicted to stably fold (ΔG = −11.6) into two symmetric stemloops identical to the largest stem loops of the full-lengthaptamer (Fig. 1C). As this minimized E3 aptamer retains thecell targeting and internalization properties of the full-lengthaptamer, yet has a length appropriate for efficient chemicalsynthesis, the truncated 36-nt E3 aptamer was utilized for allsubsequent studies.

In Vitro Characterization of E3 Aptamer Targeting to Prostate CancerCells. To verify that chemically synthesized E3 retains aptamerfunction and effectively targets a variety of prostate cancer cells,the truncated aptamer was chemically synthesized with a 5′ C6-thiol modifier and conjugated to a maleimide-DyLight 650(DL650) fluorescent dye. The approximate binding affinity andcell specificity of DL650-E3 for five different prostate cancer celllines was then determined by staining cells with increasing con-centrations of DL650-E3. Salmon sperm DNA was included as ablocking agent to prevent nonspecific oligonucleotide binding orendocytosis and ensure that aptamer accumulation in cells was aresult of specific uptake and targeting. Cells were also stainedwith increasing concentrations of DL650-labeled nonspecificcontrol aptamer, C36 [cntrl.36 (32)], an unrelated RNA thatserves as a size-matched control for RNA length.As expected, for all prostate cancer cell lines tested (Fig. 2 and

SI Appendix, Fig. S2), flow cytometry analysis of DL650-E3

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Fig. 1. Cell-Internalization SELEX against prostate cancer cells identifies theE3 aptamer, which minimizes into an aptamer of only 36 nt. (A) Cell-In-ternalization SELEX scheme for selection of 2′F pyrimidine-modified RNAaptamers that internalize into prostate cancer cells. (B) Flow cytometryanalysis of the internalization of different selection rounds into PC-3 pros-tate cancer cells. RNA pools from rounds 0, 7, and 9 of the PC-3 selectionwere transcribed with a 3′ 22-nt tail and annealed to an AF647-labeled re-verse primer. After incubating the RNA pools with PC-3 cells for 2 h, thesolutions were removed and cells washed and treated with RNase beforeflow analysis. (C) Predicted secondary structure of the minimized 36-ntversion of the E3 aptamer, generated by mfold software.

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targeting demonstrated a dose-dependent increase in aptamerassociation with cells as the concentration of aptamer increased.By contrast, very little cell staining was observed with the controlaptamer C36, even at concentrations as high as 1 μM. Analysis ofbinding via nonlinear fits of the relative mean fluorescence vs.aptamer concentration gave apparent dissociation constants (Kds)for DL650-E3 ranging from 146 to 410 nM. Thus, the synthesized

E3 truncate maintains nanomolar range, high-affinity targeting toprostate cancer cells. Importantly, E3 targets prostate cancer cellsbroadly, regardless of tumor origin or androgen sensitivity status.

Confocal Microscopy Verifies E3 Internalization into Prostate CancerCells. To verify internalization, and not merely cell-surface bind-ing, of the E3 aptamer into prostate cancer cells, E3 was synthe-sized with a 5′ C6-thiol modifier and conjugated to the fluorescentdye maleimide-Alexa Fluor 488 (AF488). Confocal microscopy of22Rv1 prostate cancer cells stained with the AF488-E3 conjugateshows intracellular accumulation of the aptamer, with punctatestaining throughout the cytoplasm (Fig. 3A). In contrast, stainingwith the control AF488-C36 resulted in minimal RNA accumu-lation in cells. Additionally, E3 partially colocalizes with a lyso-somal stain (Fig. 3B), indicating that the aptamer probably enterscells via active-targeting receptor-mediated endocytosis, resultingin endosomal to lysosomal deposition of the E3 RNA; blockingclathrin-mediated endocytosis also inhibits E3 uptake (SI Appen-dix, Fig. S3), further supporting this hypothesis. Thus, as expectedfrom the internalization-biased selection for prostate cancer spe-cific aptamers, the E3 aptamer internalizes into 22Rv1 prostatecells via an active-targeting mechanism.

E3 MMAE and MMAF Drug Conjugates Inhibit Proliferation of ProstateCancer Cells, but Not Normal Cells. The cellular targeting and in-ternalization abilities of E3 make it a potentially useful ligand fordrug delivery. As E3 appears to enter cells via an endosomal–lyosomal pathway (Fig. 3B), we chose to conjugate E3 to two dif-ferent highly toxic auristatin derivatives (Fig. 4A). The MMAEderivative is membrane permeable and able to escape from cellularcompartments whereas the similar MMAF derivative is membraneimpermeable. Comparing these two drugs allows us to probe therole of release from intracellular compartments in E3 drug delivery,an important consideration in MMAE and MMAF toxicity as bothdrugs need to reach the cytoplasm to exert their function; once inthe cytoplasm, the auristatin derivatives bind to tubulin, causingapoptosis (24, 25).We chemically conjugated the aptamer to MMAE or MMAF

using the same maleimide linkers that have been used for the clini-cally successful antibody–MMAE and –MMAF conjugates. E3 wassynthesized with a 5′C6-thiol linker for conjugation to either maleimide-caproyl-valine-citrulline-p-aminobenzylcarbamate-MMAE or tomaleimidocaproyl-MMAF, via thiol-maleimide chemistry (Fig. 4A).As free MMAE is membrane permeable, conjugation via a valine-citrulline-aminobenzylcarbamate linker inactivates the drug, preventing

Fig. 3. Confocal microscopy visualization of E3 internalization into 22Rv1 prostate cancer cells. Cells were treated for 1 h with 1 μM of either (A) AF488-E3 orAF488-C36 ± CellMask Deep Red Plasma Membrane Stain or (B) AF488-E3 + LysoTracker Deep Red stain to label acidic vesicles. After washing, Hoechst33342 was added to all samples to stain the nuclei. Fluorescence was observed on a Leica SP5 inverted confocal microscope. (White scale bars: 20 μm.)

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Fig. 2. E3 targets prostate cancer cells with nanomolar apparent affinity. Flowcytometry analysis of increasing concentrations of DL650-E3 aptamer or DL650-C36 control aptamer incubated with (A) PC-3, (B) 22Rv1, or (C) VCaP cells.Aptamers were incubated on cells for 1 h before washing cells and analyzing byflow cytometry. Binding curves represent data from three independent ex-periments, with the mean fluorescent intensity of aptamer-treated samplesnormalized to the mean fluorescent intensity of the cells alone signal.

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it from crossing cell membranes on its own, and making its cellularinternalization dependent on the conjugated targeting ligand (24).Once internalized into cells, the valine-citrulline peptide portion ofthe linker is cleaved by the lysosomal protease cathepsin B, re-leasing a p-aminobenzylcarbamate intermediate of the drug thatundergoes spontaneous fragmentation to release active MMAE.As MMAF is not cell permeable, its cell targeting is entirely de-pendent upon any attached targeting ligand and a shorter non-cleavable maleimidocaproyl linker can be used for targeting ligandattachment (25).As expected based upon the E3 aptamer’s affinity and specificity

for internalizing into prostate cancer cells, the MMAE-E3 andMMAF-E3 conjugates induced cell death in all prostate cancer cellstested, with IC50s ranging from 2.3 to 152 nM (Fig. 4 B and C and SIAppendix, Fig. S4 and Table S4). Not surprisingly, the MMAE-E3 conjugates were more efficacious than the MMAF-E3 conjugateson all prostate cancer lines, likely due to the ability of MMAE toefficiently escape into the cytoplasm upon lysosomal cleavage of theMMAE-E3 linker. The MMAF-E3 conjugates exhibited a morevariable effect on the different prostate cancer cell lines, with somecell lines appearing more sensitive to MMAF treatment (Fig. 4C andSI Appendix, Fig. S4B and Table S4). For example, while MMAF-E3 was only 1.6-fold less toxic to 22Rv1 cells than MMAE-E3,LNCaP cells were ∼26-fold less sensitive to the MMAF-E3 con-jugate. These variances may be related to differential levels ofMMAFendosomal or lysosomal escape, with some cells having “leakier” in-tracellular compartments than others.

AlthoughMMAE-E3 was more toxic thanMMAF-E3, this toxicitycame at the price of reduced specificity, as the control MMAE-C36 conjugate was significantly more toxic than the MMAF-C36 conjugate on four of the five cell lines tested (Fig. 4 B and C andSI Appendix, Fig. S4 and Table S4). Even at drug concentrations of1 μM, we failed to reach the IC50 of MMAF-C36 on the 22Rv1, DU145, LNCaP, and VCaP cell lines, lending great specificity to theMMAF-E3 conjugate versus control. The increased toxicity ofMMAE-C36 versus MMAF-C36 is not unexpected as MMAE mayrelease from the control RNA extracellularly, allowing free MMAEto enter cells on its own. Additionally, MMAE released from thelow level of internalized C36 conjugate may more efficiently escapefrom intracellular compartments to reach the cytoplasm.While the MMAF-C36 conjugate was also less toxic to PC-

3 cells than MMAE-C36, MMAF-C36 was only 12-fold less toxicthan the targeted MMAF-E3, compared with a 25-fold differ-ence in toxicity between MMAE-E3 and MMAE-C36. It isunclear why PC-3 cells are less resistant to the control MMAF-C36 conjugate than the other cell lines, although this may berelated to a difference in cell structure and origin; the PC-3 cellline is thought to derive from the more rare form of prostaticsmall-cell neuroendocrine carcinoma, compared with the typicaladenocarcinoma origin of most prostate cancers (33). Impor-tantly, neither MMAE-E3 nor MMAF-E3 exhibited specifictoxicity toward normal PrECs (Fig. 4 B and C and SI Appendix,Table S4). Although the slower growth of normal epithelial cellsmakes them inherently less susceptible to tubulin inhibitors such

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Fig. 4. E3 MMAE and MMAF drug conjugates are toxic to prostate cancer, but not normal cells. (A) Structures of E3 aptamer conjugated to maleimide-caproyl-valine-citrulline-p-aminobenzylcarbamate-MMAE or maleimidocaproyl-MMAF. (B) Viability of PC-3, 22Rv1, and VCaP prostate cancer cells or normalPrEC cells treated with MMAE-E3 and MMAE-C36 or (C) MMAF-E3 and MMAF-C36. Cells were incubated with increasing concentrations of aptamer–drugconjugates and viability determined at 144 h. Data shown are the average of three to four individual experiments. **P ≤ 0.0074, ***P ≤ 0.0003, ****P <0.0001 versus control C36 conjugates.

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as MMAE and MMAF, confocal microscopy further demon-strates that E3 does not specifically target PrEC cells (SI Ap-pendix, Fig. S5).

An Antidote to E3 Prevents Cell Targeting and Internalization andPrevents Cytotoxicity of E3–Drug Conjugates. To explore the po-tential use of antidotes to control the activity of aptamer–drugconjugates, we designed a series of 18–20-nt cDNA antidotesagainst the E3 aptamer (SI Appendix, Fig. S6–S8). Synthesizing thebest-performing DNA antidote as a 2′OMe RNA antidote resultedin complete blockage of E3-specific cell binding and internalizationas monitored by flow cytometry, bringing E3 internalization downto the same background level as the nonspecific control C36 (Fig. 5A and B), with only a 1:1 antidote:aptamer ratio required to inhibit90% of E3 targeting (SI Appendix, Fig. S9). Most importantly,twofold excess of E3 antidote 5 neutralized both the MMAF-E3 and MMAE-E3 conjugates, preventing cell killing (Fig. 5 C andD). These results demonstrate that E3 is a tunable targeting ligandwhose activity and delivery can be turned off via antidote treatmentif side effects occur despite the selectivity of E3 for cancer cells.

The E3 Aptamer Targets Prostate Tumors in Vivo and the MMAF-E3Drug Conjugate Inhibits Tumor Growth.As E3 was selected in vitro,it is important to verify that the aptamer retains prostate cancertargeting in vivo. Significantly, E3 localizes to 22Rv1 prostatexenografts in vivo, as evidenced by near-infrared (NIR) in vivoimaging of mice i.v. injected with Alexa Fluor 750 (AF750)-labeledE3 (Fig. 6A). By contrast, the control AF750-C36 conjugate did notaccumulate in the tumors. To investigate the ability of E3 to deliverdrugs and inhibit tumor growth in vivo, we chose to test MMAF-E3 against the 22Rv1 prostate cancer model, as 22Rv1 cells werethe most sensitive to MMAF-E3 in vitro while maintaining greatspecificity for MMAF-E3 compared with the control MMAF-

C36 conjugate. Additionally, the MMAF-E3 conjugate had ahalf-life of 18 h in mouse serum, compared with the less-stableMMAE-E3 conjugate with a half-life of only ∼2 h (SI Appendix, Fig.S10). Mice bearing 22Rv1 xenografts were i.v. treated with PBS orwith MMAF-E3 or control MMAF-C36 (1.03 mg/kg, based on drugconcentration). Treatment with MMAF-E3, but not with MMAF-C36, significantly inhibited tumor growth (P = 0.0324) and in-creased survival (P = 0.0163) compared with buffer-treated mice.Additionally, MMAF-E3 significantly increased survival comparedwith mice treated with the control MMAF-C36 (P = 0.0192). Thus,the E3 aptamer maintains both its prostate cancer targeting and itstherapeutic cancer cell killing effects in vivo.

DiscussionThe need for improved cancer therapies with fewer side effectshas led to the clinical development of targeted therapies such astherapeutic antibodies and ADCs. Although antibody-based can-cer therapies have proven beneficial, highlighting the ability oftargeted therapies to improve cancer patient outcomes, improveddrug-targeting strategies are needed that further focus cytotoxicagents toward cancer cells and minimize drug delivery to normalcells that express even low levels of target receptor. Aptamers areemerging as targeting agents with antibody-like affinity coupledwith the ease of chemical synthesis and modification.With the goal of developing an aptamer-targeted therapy for

prostate cancer, we used Cell-Internalization SELEX to identifythe E3 aptamer, which internalizes into prostate cancer cellswithout targeting normal prostate cells. Truncation of the E3aptamer into an easily synthesizable 36-nt 2′F-modified RNAallowed for subsequent conjugation to the highly toxic drugsMMAE and MMAF. Both the MMAE-E3 and MMAF-E3 con-jugates are toxic to prostate cancer cells, with IC50s in the nano-molar range, but do not affect normal prostate epithelial cells

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Fig. 5. An antidote oligonucleotide to E3 prevents cell targeting and in-ternalization and counteracts the cytotoxicity of E3–drug conjugates. (A)Region of the E3 aptamer targeted by the antidote oligonucleotide (repre-sented as a purple line). (B) Flow cytometry analysis of PC-3 cells incubatedwith 250 nM of DL650-C36 or with 250 nM of DL650-E3 ± 10-fold excessantidote 5. Antidote was incubated on cells for 1 h before incubating withaptamer solutions for 1 h. Cells were then washed and analyzed by flowcytometry. (C) Viability of 22Rv1 cells treated with 100 nM of MMAF-E3 orcontrol conjugate MMAF-C36 ± twofold excess of antidote 5 or with (D)100 nM of MMAE-E3 or control conjugate MMAE-C36 ± twofold excess ofantidote 5. Viability was determined 144 h post aptamer addition. Datashown are the average of four individual experiments. ***P = 0.0001 versusMMAF-E3 and ****P < 0.0001 versus MMAE-E3.

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Fig. 6. The E3 aptamer targets prostate cancer xenografts in vivo, and MMAF-E3 treatment inhibits tumor growth. s.c. 22Rv1 prostate tumors were establishedin the right flank of nu/nu mice. (A) Tumor-bearing mice were injected via tailvein with 2 nmol of either AF750-E3 or AF750-C36 (n = 4) and imaged for NIRfluorescence. Shown are representative images from 24 h post aptamer injection.(B and C) Once tumors reached at least 50 mm3, mice were treated via tail veinwith PBS or with MMAF-E3 or MMAF-C36 (1.03 mg/kg). Mice were treated q4 d × 6, starting on day 11, as indicated by the arrows. (B) Tumor growth curvesdemonstrate that MMAF-E3, but not control MMAF-C36, significantly inhibitstumor growth compared with control PBS (*P = 0.0324). (C) Kaplan–Meier sur-vival curves show a significant increase in survival for mice treated with MMAF-E3 compared with both buffer-treated and MMAF-C36–treated mice(*P = 0.0163 and *P = 0.0192, respectively). Treatment with control MMAF-C36 does not significantly change survival compared with PBS treatment.

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above a nonspecific control. An added advantage of the E3 con-jugates is their tunable toxicity: Antidote treatment abrogates thecytotoxicity of both conjugates. Importantly, MMAF-E3 maintainsits efficacy in vivo, significantly inhibiting tumor growth comparedwith control treatment. Thus, we developed a reversible aptamer–drug conjugate for the treatment of prostate cancer.The in vivo anti-tumor effects of MMAF-E3 are very promising

for an aptamer–drug conjugate expected to have an in vivo half-life of minutes (reviewed in ref. 34). Thus, optimizing the MMAF-E3 conjugate to extend in vivo circulation time should improve itsanti-tumor efficacy. Although high molecular weight PEG haspreviously been used to extend aptamer half-life for clinicalstudies (16, 19), preexisting anti-PEG antibodies caused a smallpercentage of patients (∼0.6%) to have severe allergic reactions(35). However, as PEG is commonly used in oncologic therapiesdespite its known allergic potential, it may be an acceptable way toextend aptamer half-life for the oncology setting. Alternatively, wecould explore other ways to extend aptamer half-life such asconjugation to other high molecular weight carriers.Most previously reported aptamer–drug conjugates have used

the traditional chemotherapeutic doxorubicin, via either chemicalconjugation or intercalation into the aptamer structure (reviewedin refs. 36 and 37). By contrast, only a few reports of aptamersconjugated to highly toxic agents have appeared (23, 38, 39), and,notably, MMAE-E3 appears ∼50-fold more potent than a recentlydescribed aptamer–MMAE conjugate (23). However, none of thesehighly toxic aptamer conjugates were tested in vivo. Here we reportthe development and in vivo activity of a class of targeted anticancer

therapeutics: aptamers conjugated to highly toxic chemotherapeu-tics. We demonstrate that a 1:1 direct chemical conjugate ofaptamer to the highly toxic drug MMAF kills prostate cancer cells invitro and tumors in vivo. Additionally, we report the use of antidotesto block aptamer–drug conjugate toxicity, providing a safety switchin the rare event that cytotoxicity is encountered in vivo.

Materials and MethodsAll studies were conducted in accordance with the Guide for the Care andUse of Laboratory Animals (40) and approved by the Duke University In-stitutional Animal Care and Use Committee (Protocols A086-14-04 & A076-17-03). Detailed methods are described in SI Appendix, Materials andMethods. RNA was synthesized (32) as previously described. Auristatins wereconjugated via thiol-maleimide chemistry using the linkers shown in Fig. 4A.Cell-Internalization SELEX was performed as described in Fig. 1A, flowcytometry as in Figs. 2 and 5B, confocal microscopy as in Fig. 3, cell viabilitystudies as in Figs. 4 B and C and 5 C and D, and animal studies as in Fig. 6.

ACKNOWLEDGMENTS. We thank Keith E. Maier for his assistance witholigonucleotide synthesis. This work was supported by Department ofDefense (DoD) Prostate Cancer Research Program (PCRP) PostdoctoralTraining Award PC131874 (to B.P.G.) and Synergistic Idea Award PC111812P2/W81XWH-12-1-0262 (to B.A.S.); Stand Up to Cancer Innovative Research GrantSU2C-AACR-IRG-0809 (to M.L.); and National Cancer Institute (NCI) GrantsR21CA182330 (to M.L.) and R21CA157366-03 (to M.L.). Additionally, this workwas supported by Duke Cancer Institute (DCI) NCI Grant P30-CA014236 fundingfor the Duke Optical Molecular Imaging and Analysis Facility (in vivo imaging),DCI Flow Cytometry Shared Resource (flow cytometry), and Duke LightMicroscopy Core Facility (confocal microscopy).

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