Tunable Cytotoxic Aptamer–Drug Conjugates for the Treatment of Prostate Cancer

Home , Maleimide, Monomethyl auristatin E

Tunable cytotoxic aptamer–drug conjugates for the treatment of prostate cancer

Bethany 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, NY 10461; 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 prostate While antibodies require humanization and are difficult to tumor lesions while sparing normal organ tissue are desperately chemically modify with precision, aptamers, small RNA or DNA needed. With the goal of developing an improved drug-targeting ligands, have emerged as targeting agents with antibody-like af- strategy, we turned to a new class of targeted anticancer finity (picomolar to nanomolar range) (reviewed in refs. 13–15) therapeutics: aptamers conjugated to highly toxic chemothera- and the added benefit of ease of chemical synthesis and modifi- peutics. Cell selection for aptamers with prostate cancer specificity cation. Importantly, aptamers, unless formulated with poly- yielded the E3 aptamer, which internalizes into prostate cancer ethyleneglycol (PEG) (15), have thus far proven to be nontoxic, cells without targeting normal prostate cells. Chemical conjugation even when delivered to animals and humans at high doses (16–19). of E3 to the drugs monomethyl auristatin E (MMAE) and mono- Traditionally aptamers are generated in the laboratory through a methyl auristatin F (MMAF) yields a potent cytotoxic agent that process known as Systematic Evolution of Ligands by EXponential efficiently kills prostate cancer cells in vitro but does not affect enrichment (SELEX), whereby a random oligonucleotide library normal prostate epithelial cells. Importantly, the E3 aptamer is repetitively bound with a protein target of interest (20, 21). In targets tumors in vivo and treatment with the MMAF-E3 conjugate recent years, a Cell-Internalization SELEX technique, involving a significantly inhibits prostate cancer growth in mice, demonstrat- cell target instead of a protein target, has been used to identify ing the in vivo utility of aptamer–drug conjugates. Additionally, oligonucleotides that internalize into different cell types (reviewed we report the use of antidotes to block E3 aptamer–drug conju- in ref. 22). Using two independent Cell-Internalization selections MEDICAL SCIENCES gate cytotoxicity, providing a safety switch in the unexpected against a variety of different prostate cancer cell lines, we identi- event of normal cell killing in vivo. fied an RNA aptamer, termed E3, specific for internalization into prostate cancer, but not normal prostate cells. – aptamer | aptamer–drug conjugate | prostate cancer | drug targeting | As a recent report suggests that aptamer drug conjugates that antidote control utilize highly toxic drugs can kill cells in vitro (23), we sought to develop and evaluate the in vivo activity of E3–drug conjugates to determine if this approach has clinical potential. We chemically rostate cancer is both the most commonly diagnosed malig- conjugated E3 to the auristatin drugs MMAE and monomethyl Pnancy and the second most common cause of cancer-related aruistatin F (MMAF) that have been successful in tumor targeting mortality for men in the United States (1). Therapies that antibody conjugates. Both auristatin drugs are potent microtubule eliminate both local and metastatic prostate tumor legions while inhibitors that are too toxic to be used in an unconjugated state sparing normal organ tissue are desperately needed. Recent (24, 25). The MMAE-E3 and MMAF-E3 aptamer conjugates exciting oncology advances have come from an old idea: anti- – body drug conjugates (ADCs). While the idea was first tested Significance 30 y ago (reviewed in ref. 2), it is only the development of new, highly toxic chemotherapy drugs coupled with improved antibody–drug linker chemistry that has led to Food and Drug Ad- As prostate cancer is the second leading cause of cancer-related ministration approval of ADCs such a brentuximab vedotin death among men in the United States, an unmet medical need (anti-CD30–MMAE) and ado-trastuzumab emtansine (anti-HER2– exists for therapies that eliminate prostate tumors while pre- DM1). Several ADCs are currently in clinical trials for prostate venting toxicity in normal tissue. Recently, such tumor-targeting cancer, including both a prostate-specific membrane antigen (PSMA) therapies have gained Food and Drug Administration approval in antibody (3–5) and an SCL44A4 antibody (6, 7) conjugated to the form of antibody drug conjugates. However, a need exists for new tumor-targeting therapies that are easier to manipulate and the highly toxic drug monomethyl auristatin E (MMAE). In all synthesize. By selecting for RNA ligands that internalize into these cases, the molecular target of the antibody is a known receptor prostate cancer cells but not normalprostatecells,wedeveloped up-regulated on the target cancer cell. Unfortunately almost all of the antigens that are recognized by another class of targeted agents. We demonstrate that the E3 antibodies and ADCs are not exclusively expressed on cancer RNA selectively internalizes into prostate cancer cells and that cells, but also expressed at lower levels in normal tissue (8). E3 highly toxic drug conjugates are potent anti-tumor agents. Therefore, not surprisingly, toxicities have been observed with antibody-targeted therapies, resulting, at least in part, from an- 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. tibody accumulation in normal tissue (9 12). For example, wrote the paper. HER2-targeting antibodies are associated with significant car- Conflict of interest statement: b3 bio, inc. (D.P.A. and A.P.B.) and Duke University (B.P.G. diovascular toxicities due to HER2 expression in cardiac tissue and B.A.S.) have submitted patent applications. (11), and epidermal growth factor receptor (EGFR)-targeted This article is a PNAS Direct Submission. antibodies induce significant cutaneous toxicities (9, 10) due to cutaneous expression of EGFR. Therefore, to reduce patient Published under the PNAS license. morbidity and improve the therapeutic window of targeted cy- 1Present address: Vitrisa Therapeutics, Durham, NC 27701. totoxic agents, improved drug-targeting strategies are needed 2To whom correspondence should be addressed. Email: [email protected]. that further focus cytotoxic agents toward cancer cells and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. minimize drug delivery to normal cells expressing even low levels 1073/pnas.1717705115/-/DCSupplemental. of target receptor. Published online April 16, 2018.

www.pnas.org/cgi/doi/10.1073/pnas.1717705115 PNAS | May 1, 2018 | vol. 115 | no. 18 | 4761–4766 Downloaded by guest on September 28, 2021 efficiently killed prostate cancer cells in culture, but not normal Selection for RNA Cellular Internalization A 5 prostate cells, with IC50s in the nanomolar range. 4x10 cells Incubate with We additionally aimed to exploit the unique ability of com- Library of ~1014 RNAs plementary antidote oligonucleotides to exquisitely and rapidly Normal prostate cells (R0) control aptamer activity (26, 27) so as to prevent unwanted Prostate cancer cells (R1-14) - Wash aptamer targeting to normal cells if needed, as we have observed Repeat 8-13 times - Trypsinize RNAse that these antidotes effectively turn off aptamer targeting and (Rounds 2-14) - Wash function in humans within minutes of injection (15, 28, 29). Herein, we demonstrate that E3 targeting and internalization Selection 1: PC-3 Prostate Cancer Cells (9 Rounds) can also be rapidly turned off via antidote addition. Treatment Selection 2: Toggle between LNCaP, PC-3, DU 145, 22Rv1 with antidote also neutralizes both the MMAE-E3 and MMAF Prostate Cancer Cells (14 Rounds) conjugates and prevents cell death in vitro, providing a safety switch in the rare case that our E3 conjugates were to experience the same type of unwanted accumulation in normal tissue, as has Transcribe Trizol been seen with antibody conjugates. extract Most importantly, both the E3 aptamer and MMAF-E3 drug PCR Reverse RNA amplify conjugate target and localize to prostate tumors in vivo, with Transcribe MMAF-E3 significantly inhibiting tumor growth without en- Truncated E3 Aptamer gendering obvious toxicities. Thus, these animal studies indicate B PC-3 Cell Selection C 20 CA G A that conjugation of internalizing aptamers to highly toxic che- 100 Cells G C motherapeutic agents yields a class of targeted anticancer ther- Round 0 C A 80 Round 7 U U apeutics, and the MMAF-E3 conjugate represents a targeted Round 9 U C U A approach to treat patients with prostate cancer. 60 C G

10 G C

G C

Results % of Max 40 G C 30 Selection of an RNA Aptamer That Internalizes into Prostate Cancer C C 20 U U Cells. U C In an unbiased approach to identify RNA aptamers that U A

0 C G internalize into prostate cancer cells at various stages of malig- 2 3 4 5 010 10 10 10 G C nancy, we performed two Cell-Internalization SELEX using a 2′ Alexa Fluor 647 5’ G C 3’ fluoro pyrimidine modified (30) RNA library comprising a mod- 14 ified, nuclease-resistant RNA pool of ∼10 different RNAs. Two Fig. 1. Cell-Internalization SELEX against prostate cancer cells identifies the different selection strategies were employed. In the first, positive E3 aptamer, which minimizes into an aptamer of only 36 nt. (A) Cell-In- selections targeting PC-3 prostate cancer cells were combined with ternalization SELEX scheme for selection of 2′F pyrimidine-modified RNA a strong negative selection against normal prostate epithelial cells aptamers that internalize into prostate cancer cells. (B) Flow cytometry (PrEC) to deplete RNA molecules capable of internalizing into analysis of the internalization of different selection rounds into PC-3 pros- noncancerous cells. In the second, the cell target for the positive tate cancer cells. RNA pools from rounds 0, 7, and 9 of the PC-3 selection selection was varied and included toggling between the LNCaP, were transcribed with a 3′ 22-nt tail and annealed to an AF647-labeled re- PC-3, DU 145, and 22Rv1 cell lines (Fig. 1A and SI Appendix, verse primer. After incubating the RNA pools with PC-3 cells for 2 h, the Tables S1 and S2). These four prostate cancer cell lines represent solutions were removed and cells washed and treated with RNase before different stages of prostate cancer progression, ranging from high flow analysis. (C) Predicted secondary structure of the minimized 36-nt sensitivity and dependence upon androgen hormones (LNCaP version of the E3 aptamer, generated by mfold software. cells), to low androgen sensitivity (22Rv1 cells), to androgen in- dependence (PC-3 cells), and finally to a brain metastasis de- The 83-nt-long E3 aptamer folds into two predicted secondary rivative (DU 145). Additionally, to exclude PSMA as a target, we SI Appendix ′ ′ chose to select against both PSMA-positive (LNCaP and 22Rv1) structures ( , Fig. S1). Truncation from the 5 and 3 ends of the RNA results in a 36-nt minimized E3 structure that is and PSMA-negative (PC-3 and DU 145) cell lines. Here again, ΔG = − positive rounds of selection targeting these cells were combined predicted to stably fold ( 11.6) into two symmetric stem loops identical to the largest stem loops of the full-length with a strong negative selection against PrECs. For both selec- C tions, after incubating cells with the library of RNA variants and aptamer (Fig. 1 ). As this minimized E3 aptamer retains the washing away nonbinding aptamers, cell-surface-bound RNA was cell targeting and internalization properties of the full-length degraded via treatment with a harsh RNase mixture that degrades aptamer, yet has a length appropriate for efficient chemical 2′fluoro modified RNA (31), leaving only RNAs that had in- synthesis, the truncated 36-nt E3 aptamer was utilized for all ternalized into cells. Subsequent reverse transcription, PCR am- subsequent studies. plification, and transcription of the internalized RNA completed a round of internalization selection. In Vitro Characterization of E3 Aptamer Targeting to Prostate Cancer To assess selection progress, RNA pools from different se- Cells. To verify that chemically synthesized E3 retains aptamer lection rounds were transcribed with a 3′ 22-nt tail that annealed function and effectively targets a variety of prostate cancer cells, ′ to an Alexa Fluor 647-labeled reverse primer. Subsequent flow the truncated aptamer was chemically synthesized with a 5 C6- cytometry analysis clearly demonstrated an increase of in- thiol modifier and conjugated to a maleimide-DyLight 650 ternalizing aptamers as the selection progressed through addi- (DL650) fluorescent dye. The approximate binding affinity and tional rounds, as shown for the PC-3 cell selection (Fig. 1B). cell specificity of DL650-E3 for five different prostate cancer cell After nine rounds of selection against PC-3 cells, 12 unique lines was then determined by staining cells with increasing con- RNAs were evaluated; two of them, the E3 and A3 aptamers, centrations of DL650-E3. Salmon sperm DNA was included as a were found to internalize (SI Appendix, Table S3). Interestingly, blocking agent to prevent nonspecific oligonucleotide binding or after 14 rounds of toggling between LNCaP, PC-3, DU 145, and endocytosis and ensure that aptamer accumulation in cells was a 22Rv1 cells, the E3 and A3 aptamers also emerged as well as one result of specific uptake and targeting. Cells were also stained additional internalizing aptamer (D11, SI Appendix, Table S3). with increasing concentrations of DL650-labeled nonspecific Unexpectedly, the E3, A3, and D11 aptamers all competed for control aptamer, C36 [cntrl.36 (32)], an unrelated RNA that cellular binding and internalization, with the E3 aptamer con- serves as a size-matched control for RNA length. sistently reaching the highest levels of cellular internalization. As expected, for all prostate cancer cell lines tested (Fig. 2 and Therefore, the E3 aptamer was chosen for further studies. SI Appendix,Fig.S2), flow cytometry analysis of DL650-E3

4762 | www.pnas.org/cgi/doi/10.1073/pnas.1717705115 Powell Gray et al. Downloaded by guest on September 28, 2021 A PC-3 Cells E3 truncate maintains nanomolar range, high-affinity targeting to DL650-E3 DL650-C36 Kdapp= 146 ± 28.0 nM prostate cancer cells. Importantly, E3 targets prostate cancer cells 100 100 60 DL650-E3 Cells DL650-C36 broadly, regardless of tumor origin or androgen sensitivity status. 80 80 50nM 100nM 40 60 60 250nM Confocal Microscopy Verifies E3 Internalization into Prostate Cancer 500nM 40 40 1000nM 20 Cells. % of Max To verify internalization, and not merely cell-surface bind-

% of Max Relative MFI 20 20 ing, of the E3 aptamer into prostate cancer cells, E3 was synthe- 0 ′ 0 0 0 250 500 750 1000 sized with a 5 C6-thiol modifier and conjugated to the fluorescent 3 3 4 5 3 3 4 5 -10 0 10 10 10 -10 0 10 10 10 [Aptamer] (nM) DyLight 650 DyLight 650 dye maleimide-Alexa Fluor 488 (AF488). Confocal microscopy of B 22RV1 Cells 22Rv1 prostate cancer cells stained with the AF488-E3 conjugate DL650-E3 DL650-C36 Kdapp= 204 ± 91.4 nM shows intracellular accumulation of the aptamer, with punctate 100 100 Cells 100 staining throughout the cytoplasm (Fig. 3A). In contrast, staining 50nM DL650-E3 80 80 100nM 80 DL650-C36 with the control AF488-C36 resulted in minimal RNA accumu- 60 60 250nM 60 500nM lation in cells. Additionally, E3 partially colocalizes with a lyso-

% of Max 40 % of Max 40 1000nM 40 somal stain (Fig. 3B), indicating that the aptamer probably enters

Relative MFI 20 20 20 cells via active-targeting receptor-mediated endocytosis, resulting 0 in endosomal to lysosomal deposition of the E3 RNA; blocking 0 0 0 250 500 750 1000 3 3 4 5 3 3 4 5 SI Appen- -10 0 10 10 10 -10 0 10 10 10 [Aptamer] (nM) clathrin-mediated endocytosis also inhibits E3 uptake ( DyLight 650 DyLight 650 dix VCaP Cells ,Fig.S3), further supporting this hypothesis. Thus, as expected C from the internalization-biased selection for prostate cancer spe- DL650-E3 DL650-C36 Kdapp= 358 ± 100 nM 20 cific aptamers, the E3 aptamer internalizes into 22Rv1 prostate 100 100 Cells DL650-E3 50nM DL650-C36 cells via an active-targeting mechanism. 80 80 100nM 15 60 60 250nM 500nM 10 E3 MMAE and MMAF Drug Conjugates Inhibit Proliferation of Prostate 40 40 1000nM

% of Max % of Max 5 Cancer Cells, but Not Normal Cells. The cellular targeting and in- 20 20 Relative MFI ternalization abilities of E3 make it a potentially useful ligand for 0 0 0 0 250 500 750 1000 drug delivery. As E3 appears to enter cells via an endosomal– -103 0 103 104 105 3 3 4 5 -10 0 10 10 10 [Aptamer] (nM) DyLight 650 DyLight 650 lyosomal pathway (Fig. 3B), we chose to conjugate E3 to two different highly toxic auristatin derivatives (Fig. 4A). The MMAE Fig. 2. E3 targets prostate cancer cells with nanomolar apparent affinity. Flow derivative is membrane permeable and able to escape from cellular MEDICAL SCIENCES cytometry analysis of increasing concentrations of DL650-E3 aptamer or DL650- compartments whereas the similar MMAF derivative is membrane C36 control aptamer incubated with (A)PC-3,(B)22Rv1,or(C)VCaPcells. impermeable. Comparing these two drugs allows us to probe the Aptamers were incubated on cells for 1 h before washing cells and analyzing by role of release from intracellular compartments in E3 drug delivery, flow cytometry. Binding curves represent data from three independent ex- an important consideration in MMAE and MMAF toxicity as both periments, with the mean fluorescent intensity of aptamer-treated samples drugs need to reach the cytoplasm to exert their function; once in normalized to the mean fluorescent intensity of the cells alone signal. the cytoplasm, the auristatin derivatives bind to tubulin, causing apoptosis (24, 25). targeting demonstrated a dose-dependent increase in aptamer We chemically conjugated the aptamer to MMAE or MMAF using the same maleimide linkers that have been used for the clini- association with cells as the concentration of aptamer increased. cally successful antibody–MMAE and –MMAF conjugates. E3 was By contrast, very little cell staining was observed with the control ′ μ synthesized with a 5 C6-thiol linker for conjugation to either maleimide- aptamer C36, even at concentrations as high as 1 M. Analysis of caproyl-valine-citrulline-p-aminobenzylcarbamate-MMAE or to binding via nonlinear fits of the relative mean fluorescence vs. maleimidocaproyl-MMAF, via thiol-maleimide chemistry (Fig. 4A). aptamer concentration gave apparent dissociation constants (Kds) As free MMAE is membrane permeable, conjugation via a valine- for DL650-E3 ranging from 146 to 410 nM. Thus, the synthesized 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 or AF488-C36 ± CellMask Deep Red Plasma Membrane Stain or (B) AF488-E3 + LysoTracker Deep Red stain to label acidic vesicles. After washing, Hoechst 33342 was added to all samples to stain the nuclei. Fluorescence was observed on a Leica SP5 inverted confocal microscope. (White scale bars: 20 μm.)

Powell Gray et al. PNAS | May 1, 2018 | vol. 115 | no. 18 | 4763 Downloaded by guest on September 28, 2021 Thiol Reactive Val-Cit PABC MMAE A Maleimide (cathepsin cleavage) (linker) (membrane permeable)

O O OH O O NH N O O O N N H N E3 Aptamer O NH N H O O O N N H H O O

Thiol Reactive O NH2 MMAF Maleimide (membrane impermeable)

O O O O NH N N N N H E3 Aptamer N O O O H O COOH O B PC-3 Cells 22Rv1 Cells PrEC Cells

100 100 100 Cells Alone Cells Alone Cells Alone **** MMAE-E3 MMAE-E3 MMAE-E3 MMAE-C36 MMAE-C36 50 MMAE-C36 50 50 **** **** *** % Viable Cells % Viable % Viable Cells % Viable **** **** **** Cells % Viable 0 **** **** **** 0 0 0.1 1 10 100 1000 0.1 1 10 100 1000 0.1 1 10 100 1000 [MMAE] (nM) [MMAE] (nM) [MMAE] (nM) C PC-3 Cells 22Rv1 Cells PrEC Cells

100 100 Cells Alone 100 Cells Alone MMAF-E3 Cells Alone ** MMAF-E3 MMAF-C36 MMAF-E3 **** MMAF-C36 50 50 MMAF-C36 50 ** **** **** **** **** *** ** **** ** % Viable Cells % Viable % Viable Cells % Viable 0 % Viable Cells % Viable 0 0 0.1 1 10 100 1000 0.1 1 10 100 1000 0.1 1 10 100 1000 [MMAF] (nM) [MMAF] (nM) [MMAF] (nM)

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 normal PrEC cells treated with MMAE-E3 and MMAE-C36 or (C) MMAF-E3 and MMAF-C36. Cells were incubated with increasing concentrations of aptamer–drug conjugates 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.

it from crossing cell membranes on its own, and making its cellular Although MMAE-E3 was more toxic than MMAF-E3, this toxicity internalization dependent on the conjugated targeting ligand (24). came at the price of reduced specificity, as the control MMAE- Once internalized into cells, the valine-citrulline peptide portion of C36 conjugate was significantly more toxic than the MMAF- the linker is cleaved by the lysosomal protease cathepsin B, re- C36 conjugate on four of the five cell lines tested (Fig. 4 B and C and leasing a p-aminobenzylcarbamate intermediate of the drug that SI Appendix,Fig.S4andTableS4). Even at drug concentrations of undergoes spontaneous fragmentation to release active MMAE. 1 μM, we failed to reach the IC50 of MMAF-C36 on the 22Rv1, DU As MMAF is not cell permeable, its cell targeting is entirely de- 145, LNCaP, and VCaP cell lines, lending great specificity to the pendent upon any attached targeting ligand and a shorter non- MMAF-E3 conjugate versus control. The increased toxicity of cleavable maleimidocaproyl linker can be used for targeting ligand MMAE-C36 versus MMAF-C36 is not unexpected as MMAE may attachment (25). release from the control RNA extracellularly, allowing free MMAE As expected based upon the E3 aptamer’s affinity and specificity to enter cells on its own. Additionally, MMAE released from the for internalizing into prostate cancer cells, the MMAE-E3 and low level of internalized C36 conjugate may more efficiently escape MMAF-E3 conjugates induced cell death in all prostate cancer cells from intracellular compartments to reach the cytoplasm. tested, with IC50s ranging from 2.3 to 152 nM (Fig. 4 B and C and SI While the MMAF-C36 conjugate was also less toxic to PC- Appendix,Fig.S4andTableS4). Not surprisingly, the MMAE- 3 cells than MMAE-C36, MMAF-C36 was only 12-fold less toxic E3 conjugates were more efficacious than the MMAF-E3 conjugates than the targeted MMAF-E3, compared with a 25-fold differ- on all prostate cancer lines, likelyduetotheabilityofMMAEto ence in toxicity between MMAE-E3 and MMAE-C36. It is efficiently escape into the cytoplasm upon lysosomal cleavage of the unclear why PC-3 cells are less resistant to the control MMAF- MMAE-E3 linker. The MMAF-E3 conjugates exhibited a more C36 conjugate than the other cell lines, although this may be variable effect on the different prostate cancer cell lines, with some related to a difference in cell structure and origin; the PC-3 cell cell lines appearing more sensitive to MMAF treatment (Fig. 4C and line is thought to derive from the more rare form of prostatic SI Appendix,Fig.S4B and Table S4). For example, while MMAF- small-cell neuroendocrine carcinoma, compared with the typical E3 was only 1.6-fold less toxic to 22Rv1 cells than MMAE-E3, adenocarcinoma origin of most prostate cancers (33). Impor- LNCaP cells were ∼26-fold less sensitive to the MMAF-E3 con- tantly, neither MMAE-E3 nor MMAF-E3 exhibited specific jugate. These variances may be related to differential levels of MMAF toxicity toward normal PrECs (Fig. 4 B and C and SI Appendix, endosomal or lysosomal escape, with some cells having “leakier” in- Table S4). Although the slower growth of normal epithelial cells tracellular compartments than others. makes them inherently less susceptible to tubulin inhibitors such

4764 | www.pnas.org/cgi/doi/10.1073/pnas.1717705115 Powell Gray et al. Downloaded by guest on September 28, 2021 C36 conjugate. Additionally, the MMAF-E3 conjugate had a A 20 B PC-3 Cells CA G A half-life of 18 h in mouse serum, compared with the less-stable G C 100 100 C A ∼ SI Appendix Cells MMAE-E3 conjugate with a half-life of only 2h( ,Fig. U U 80 Cells U C 80 E3 U A E3 S10). Mice bearing 22Rv1 xenografts were i.v. treated with PBS or C G C36 E3+A5 with MMAF-E3 or control MMAF-C36 (1.03 mg/kg, based on drug 10 G C 60 60 G C concentration). Treatment with MMAF-E3, but not with MMAF- G C 30 40

% of Max 40 C C % of Max C36, significantly inhibited tumor growth (P = 0.0324) and in- U U U C P = U A 20 20 creased survival ( 0.0163) compared with buffer-treated mice. C G Additionally, MMAF-E3 significantly increased survival compared G C 0 0 G C P = -103 0 103 104 105 -103 0 103 104 105 with mice treated with the control MMAF-C36 ( 0.0192). Thus, DyLight 650 DyLight 650 the E3 aptamer maintains both its prostate cancer targeting and its C 22Rv1 Cells D 22Rv1 Cells therapeutic cancer cell killing effects in vivo. *** 100 100 Discussion **** The need for improved cancer therapies with fewer side effects

50 50 has led to the clinical development of targeted therapies such as therapeutic antibodies and ADCs. Although antibody-based can-

% Viable Cells % Viable Cells % Viable cer therapies have proven beneficial, highlighting the ability of 0 0 targeted therapies to improve cancer patient outcomes, improved A5 A5 drug-targeting strategies are needed that further focus cytotoxic Alone agents toward cancer cells and minimize drug delivery to normal MMAF-E3 MMAE-E3 Cells Alone MMAF-C36 Cells MMAE-C36 cells that express even low levels of target receptor. Aptamers are MMAF-E3 + A5 MMAE-E3 + MMAF-C36 + A5 MMAE-C36 + emerging as targeting agents with antibody-like affinity coupled with the ease of chemical synthesis and modification. Fig. 5. An antidote oligonucleotide to E3 prevents cell targeting and internalization and counteracts the cytotoxicity of E3–drug conjugates. (A) With the goal of developing an aptamer-targeted therapy for Region of the E3 aptamer targeted by the antidote oligonucleotide (repre- prostate cancer, we used Cell-Internalization SELEX to identify sented as a purple line). (B) Flow cytometry analysis of PC-3 cells incubated the E3 aptamer, which internalizes into prostate cancer cells with 250 nM of DL650-C36 or with 250 nM of DL650-E3 ± 10-fold excess without targeting normal prostate cells. Truncation of the E3 antidote 5. Antidote was incubated on cells for 1 h before incubating with aptamer into an easily synthesizable 36-nt 2′F-modified RNA MEDICAL SCIENCES aptamer solutions for 1 h. Cells were then washed and analyzed by flow allowed for subsequent conjugation to the highly toxic drugs cytometry. (C) Viability of 22Rv1 cells treated with 100 nM of MMAF-E3 or MMAE and MMAF. Both the MMAE-E3 and MMAF-E3 con- ± control conjugate MMAF-C36 twofold excess of antidote 5 or with (D) jugates are toxic to prostate cancer cells, with IC50s in the nano- 100 nM of MMAE-E3 or control conjugate MMAE-C36 ± twofold excess of molar range, but do not affect normal prostate epithelial cells antidote 5. Viability was determined 144 h post aptamer addition. Data shown are the average of four individual experiments. ***P = 0.0001 versus MMAF-E3 and ****P < 0.0001 versus MMAE-E3. A 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 and Prevents Cytotoxicity of E3–Drug Conjugates. To explore the potential use of antidotes to control the activity of aptamer–drug conjugates, we designed a series of 18–20-nt cDNA antidotes against the E3 aptamer (SI Appendix,Fig.S6–S8). Synthesizing the best-performing DNA antidote as a 2′OMe RNA antidote resulted in complete blockage of E3-specific cell binding and internalization BC as monitored by flow cytometry, bringing E3 internalization down to the same background level as the nonspecific control C36 (Fig. 5 A and B), with only a 1:1 antidote:aptamer ratio required to inhibit 90% 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 and D). These results demonstrate that E3 is a tunable targeting ligand whose activity and delivery can be turned off via antidote treatment if side effects occur despite the selectivity of E3 for cancer cells. Fig. 6. The E3 aptamer targets prostate cancer xenografts in vivo, and MMAF- The E3 Aptamer Targets Prostate Tumors in Vivo and the MMAF-E3 E3 treatment inhibits tumor growth. s.c. 22Rv1 prostate tumors were established Drug Conjugate Inhibits Tumor Growth. As E3 was selected in vitro, in the right flank of nu/nu mice. (A) Tumor-bearing mice were injected via tail = it is important to verify that the aptamer retains prostate cancer veinwith2nmolofeitherAF750-E3orAF750-C36(n 4) and imaged for NIR targeting in vivo. Significantly, E3 localizes to 22Rv1 prostate fluorescence. 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 vein xenografts in vivo, as evidenced by near-infrared (NIR) in vivo with PBS or with MMAF-E3 or MMAF-C36 (1.03 mg/kg). Mice were treated q imaging of mice i.v. injected with Alexa Fluor 750 (AF750)-labeled × A 4d 6, starting on day 11, as indicated by the arrows. (B) Tumor growth curves E3 (Fig. 6 ). By contrast, the control AF750-C36 conjugate did not demonstrate that MMAF-E3, but not control MMAF-C36, significantly inhibits accumulate in the tumors. To investigate the ability of E3 to deliver tumor growth compared with control PBS (*P = 0.0324). (C)Kaplan–Meier sur- drugs and inhibit tumor growth in vivo, we chose to test MMAF- vival curves show a significant increase in survival for mice treated with MMAF- E3 against the 22Rv1 prostate cancer model, as 22Rv1 cells were E3 compared with both buffer-treated and MMAF-C36–treated mice the most sensitive to MMAF-E3 in vitro while maintaining great (*P = 0.0163 and *P = 0.0192, respectively). Treatment with control MMAF- specificity for MMAF-E3 compared with the control MMAF- C36 does not significantly change survival compared with PBS treatment.

Powell Gray et al. PNAS | May 1, 2018 | vol. 115 | no. 18 | 4765 Downloaded by guest on September 28, 2021 above a nonspecific control. An added advantage of the E3 con- therapeutics: aptamers conjugated to highly toxic chemotherapeu- jugates is their tunable toxicity: Antidote treatment abrogates the tics. We demonstrate that a 1:1 direct chemical conjugate of cytotoxicity of both conjugates. Importantly, MMAF-E3 maintains aptamer to the highly toxic drug MMAF kills prostate cancer cells in its efficacy in vivo, significantly inhibiting tumor growth compared vitro and tumors in vivo. Additionally, we report the use of antidotes with control treatment. Thus, we developed a reversible aptamer– to block aptamer–drug conjugate toxicity, providing a safety switch drug conjugate for the treatment of prostate cancer. in the rare event that cytotoxicity is encountered in vivo. The in vivo anti-tumor effects of MMAF-E3 are very promising for an aptamer–drug conjugate expected to have an in vivo half- Materials and Methods life of minutes (reviewed in ref. 34). Thus, optimizing the MMAF- All studies were conducted in accordance with the Guide for the Care and E3 conjugate to extend in vivo circulation time should improve its Use of Laboratory Animals (40) and approved by the Duke University In- anti-tumor efficacy. Although high molecular weight PEG has stitutional Animal Care and Use Committee (Protocols A086-14-04 & A076- previously been used to extend aptamer half-life for clinical 17-03). Detailed methods are described in SI Appendix, Materials and studies (16, 19), preexisting anti-PEG antibodies caused a small ∼ Methods. RNA was synthesized (32) as previously described. Auristatins were percentage of patients ( 0.6%) to have severe allergic reactions conjugated via thiol-maleimide chemistry using the linkers shown in Fig. 4A. (35). However, as PEG is commonly used in oncologic therapies Cell-Internalization SELEX was performed as described in Fig. 1A, flow despite its known allergic potential, it may be an acceptable way to cytometry as in Figs. 2 and 5B, confocal microscopy as in Fig. 3, cell viability extend aptamer half-life for the oncology setting. Alternatively, we studies as in Figs. 4 B and C and 5 C and D, and animal studies as in Fig. 6. could explore other ways to extend aptamer half-life such as conjugation to other high molecular weight carriers. ACKNOWLEDGMENTS. We thank Keith E. Maier for his assistance with Most previously reported aptamer–drug conjugates have used oligonucleotide synthesis. This work was supported by Department of the traditional chemotherapeutic doxorubicin, via either chemical Defense (DoD) Prostate Cancer Research Program (PCRP) Postdoctoral conjugation or intercalation into the aptamer structure (reviewed Training Award PC131874 (to B.P.G.) and Synergistic Idea Award PC111812P2/ in refs. 36 and 37). By contrast, only a few reports of aptamers W81XWH-12-1-0262 (to B.A.S.); Stand Up to Cancer Innovative Research Grant conjugated to highly toxic agents have appeared (23, 38, 39), and, SU2C-AACR-IRG-0809 (to M.L.); and National Cancer Institute (NCI) Grants notably, MMAE-E3 appears ∼50-fold more potent than a recently R21CA182330 (to M.L.) and R21CA157366-03 (to M.L.). Additionally, this work – was supported by Duke Cancer Institute (DCI) NCI Grant P30-CA014236 funding described aptamer MMAE conjugate (23). However, none of these for the Duke Optical Molecular Imaging and Analysis Facility (in vivo imaging), highly toxic aptamer conjugates were tested in vivo. Here we report DCI Flow Cytometry Shared Resource (flow cytometry), and Duke Light the development and in vivo activity of a class of targeted anticancer Microscopy Core Facility (confocal microscopy).

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