hsp90 inhibitor–sn-38 conjugate strategy for targeted delivery of

Small Molecule Therapeutics

HSP90 Inhibitor–SN-38 Conjugate Strategy forTargeted Delivery of Topoisomerase I Inhibitor toTumorsDavid A. Proia1, Donald L. Smith1, Junyi Zhang1, John-Paul Jimenez1, Jim Sang1,Luisa Shin Ogawa1, Manuel Sequeira1, Jaime Acquaviva1, Suqin He1, Chaohua Zhang1,Vladimir Khazak2, Igor Astsaturov3, Takayo Inoue1, Noriaki Tatsuta1, Sami Osman1,Richard C. Bates1, Dinesh Chimmanamada1, and Weiwen Ying1

Abstract

The clinical benefits of chemotherapy are commonly offset byinsufficient drug exposures, narrow safety margins, and/or sys-temic toxicities.Over recent decades, a numberof conjugate-basedtargeting approaches designed to overcome these limitations havebeen explored. Here, we report on an innovative strategy thatutilizes HSP90 inhibitor–drug conjugates (HDC) for directedtumor targeting of chemotherapeutic agents. STA-12-8666 is anHDC that comprises anHSP90 inhibitor fused to SN-38, the activemetabolite of irinotecan. Mechanistic analyses in vitro establishedthat high-affinity HSP90 binding conferred by the inhibitorbackbone could be exploited for conjugate accumulation withintumor cells. In vivo modeling showed that the HSP90 inhibitor

moiety was required for selective retention of STA-12-8666, andthis enrichmentpromoted extended release of active SN-38withinthe tumor compartment. Indeed, controlled intratumoral payloadrelease by STA-12-8666 contributed to a broad therapeutic win-dow, sustained biomarker activity, and remarkable degree ofefficacy and durability of response in multiple cell line andpatient-derived xenograft models. Overall, STA-12-8666 has beendeveloped as a unique HDC agent that employs a distinct mech-anism of targeted drug delivery to achieve potent and sustainedantitumor effects. These findings identify STA-12-8666 as a prom-ising new candidate for evaluation as novel anticancer therapeu-tic. Mol Cancer Ther; 14(11); 1–11. �2015 AACR.

IntroductionChemotherapeutic drugs have been a mainstay of cancer ther-

apy for many decades; however, their effectiveness is often ham-pered by systemic toxicities due to adverse effects on normaltissues. This lack of discrimination limits their administrabledosing in the clinic, resulting in narrow therapeutic windowsand less-than-optimal efficacies. Moreover, multidrug combina-tions that represent the standard-of-care treatment modality for avariety of cancer types typically require further dose reductions tomaintain acceptable tolerability (1). Thus, targeted delivery ofcytocidal amounts of toxic agents to tumor cells remains anongoing challenge for cancer therapy. To address this concern,a variety of site-selective drug delivery strategies have beendesigned to deliver chemotherapeutics more directly to tumors(2), including coupling anticancer drugs to monoclonal antibo-dies, peptides, and synthetic polymers (3–7). In particular, sig-

nificant translational progress has been made in the field ofantibody–drug conjugates (ADC; ref. 3), evident by the recentapproval of brentuximab vedotin and ado-trastuzumab emtan-sine (4). Such conjugate-based drug delivery systems share com-monality with respect to their functional construction, compris-ing a tumor recognition moiety and cytotoxic payload connectedvia chemical linkage.

We applied these fundamentals to establish a newdrug deliverysystem termed HSP90 inhibitor–drug conjugates, or HDC, basedon the property that small-molecule inhibitors of HSP90 arepreferentially retained in tumor cells in contrast to their rapidclearance from the circulation and normal tissues (8–12). HSP90is a highly conserved molecular chaperone that plays an indis-pensable role in regulating thematuration and functional stabilityof a vast repertoire of cellular client proteins (13). HSP90 isfrequently overexpressed in tumor tissues and, importantly, existswithin cancer cells in an activated configuration that displaysgreater affinity for selective inhibitors than the resident poolfound in normal cells (14). Relative to normal cells, the rate ofHSP90 chaperone cycling accelerates in tumor cells in part due toincreased SUMOylation,which recruits co-chaperones required tomaximally stimulate HSP90 ATPase activity (15). Importantly,SUMOylation of HSP90 also facilitates the binding of ATP com-petitive inhibitors to HSP90, helping to explain the affinity ofthese agents for tumor-associated HSP90. By attaching potentchemotherapeutic drugs to HSP90 inhibitor backbones, HDCtechnology exploits this inherent retention property to moreefficiently deliver cytotoxic payloads directly into tumor tissuesand provide extended drug residency periods.

1Synta Pharmaceuticals Corp., Lexington, Massachusetts. 2Nexus-Pharma Inc., Langhorne, Pennsylvania. 3Program in Molecular Thera-peutics, Fox Chase Cancer Center, Philadelphia, Pennsylvania.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Authors: Weiwen Ying, Synta Pharmaceuticals Corp., 45 Hart-well Avenue, Lexington, MA 02421. Phone: 781-541-7243; Fax: 781-274-8228;E-mail: [email protected]; and Dinesh Chimmanamada, E-mail:[email protected].

doi: 10.1158/1535-7163.MCT-15-0455

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

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Here we report on the preclinical characterization of aprototypical HDC compound, STA-12-8666, comprised of anHSP90 inhibitor fused to SN-38, a potent topoisomeraseI inhibitor and pharmacologically active metabolite of irino-tecan (16). These proof-of-concept findings, including thedemonstration of extended intratumoral drug exposures andsuperior therapeutic indices over irinotecan therapy alone, areof considerable translational relevance for the future develop-ment of this novel class of investigational agents as cancertherapeutics.

Materials and MethodsCell lines, antibodies, and reagents

The H1975, SK-NEP-1, and SW780 cell lines were obtainedfrom ATCC between March and November 2013; the MDA-MB-231 line was purchased in October 2007. All were authenticatedby routine company DNA typing, maintained according to thesuppliers' instructions, and never continuously cultured for morethan 3 months. No further cell line authentication has beenperformed by the authors. The human small-cell lung cancer(SCLC)cell lineSR2was a gift fromDr.NiramolSavaraj (UniversityofMiami, FL) in January 2011. No authentication of the gifted linewas carried out by the authors. All antibodies were purchased fromCell Signaling Technology with the exception of the HSP70 (EnzoLife Sciences), KAP1 and phospho-KAP1 (Abcam), and GAPDH(Santa Cruz Biotechnology Inc.). Irinotecan was purchased fromLC Laboratories. Ganetespib, STA-12-8666, STA-12-9432, STA-12-8663, STA-12-9455, and STA-12-9467 were synthesized by SyntaPharmaceuticals Corp.

Immunofluorescent stainingH1975 cells were treated with vehicle (DMSO), or 1 mmol/L

ganetespib, SN-38, or STA-12-8666 for 1 hour. Following drugwashout, cells were cultured for an additional hour prior tofixation and permeabilization. Cells were stained with p-KAP1antibody (1/1000 in PBS þ 0.1% Triton X-100) overnight at 4�Cbefore incubationwithAlexa Fluor 594GoatAnti-Rabbit IgG (LifeTechnologies) for 1 hour at room temperature. Slides weremounted using DAPI-containing medium (VECTASHIELD) andimages obtained with an EVOS-FL fluorescent microscope (LifeTechnologies).

Western blottingFollowing in vitro drug assays with SN-38, ganetespib, STA-12-

9432, or STA-12-8666, H1975 tumor cells were disrupted in lysisbuffer (Cell Signaling Technology) on ice for 10 minutes. Lysateswere clarified by centrifugation and equal amounts of proteinsresolved by SDS-PAGE before transfer to nitrocellulose mem-branes (Invitrogen). Membranes were blocked with StartingBlockT20blocking buffer (ThermoScientific) and immunoblottedwiththe indicated antibodies. Antibody–antigen complexes were visu-alized using an Odyssey system (LI-COR).

In vivo xenograft tumor modelsFemale CB.17 (SCID) mice (Charles River Laboratories) at 7 to

12 weeks of age were maintained in a pathogen-free environmentand all in vivoprocedures were performed in strict accordancewiththe NIH Guide for the Care and Use of Laboratory Animals andapproved by the Synta Pharmaceuticals Corp. Institutional Ani-mal Care and Use Committee. H1975, MDA-MB-231, SK-NEP-1,SW780 (all 5� 106), or SR2 cells (7.5� 106)were subcutaneously

implanted into SCIDmice. Mice bearing established tumors wereallocated into treatment groups of 6 to 8 exhibiting similaraverage tumor volumes (100–200mm3) and intravenously dosedwith vehicle, STA-12-9432, STA-12-8666, and/or irinotecan,using the doses and schedules indicated. For the large tumorH1975 study, xenograft tumors were permitted to grow to anaverage volume of 750 mm3 (upper limit, 900 mm3) beforeinitiation of therapy. In the combination study, mice bearingSR2 tumors (n¼ 8/group) were dosed with vehicle, STA-12-8666(150 mg/kg), ganetespib (100 mg/kg), irinotecan (50 mg/kg), orthe combination of irinotecan plus ganetespib, once weekly for atotal of 4 doses. All compounds were formulated in DRD [10%dimethyl sulfoxide (DMSO), 18% Cremophor RH 40, 3.6%dextrose].

For the extended patient-derived xenograft (PDX) study, anon-ymous surgical pancreatic tumor tissues were procured at FoxChaseCancer Center under an IRB-approved protocol after receiv-ing informed patient consent. DMSO-cryopreserved PDX tumorsfrom passage 3 were subcutaneously implanted into both flanksof fifteen 5- to 8-week-old ICR-C.B17-scid mice. When tumorsreached an average 250 mm3, mice (n ¼ 5 per group) wererandomized to receive weekly dosing with vehicle [20% Cremo-phor RH 40/80%D5W (dextrose 5% in water)], irinotecan(50 mg/kg), or STA-12-8666 (150 mg/kg). Following an initial3-week treatment cycle, drug treatment was discontinued andanimals monitored for recurrence out to 150 days. Upon pro-gression, tumors were re-treated with 150 mg/kg STA-12-8666using a second weekly dosing schedule. Tumor growth inhibitionwas determined as described previously (17). Statistical analyseswere conducted using two-way ANOVA followed by Bonferronipost tests.

BioanalysisSTA-12-8666 (150 mg/kg) and irinotecan (50 mg/kg) were

administered as a single bolus injection to MDA-MB-231 xeno-graft-bearing mice (n ¼ 3). Tumor samples were collected fromeuthanized animals at 0.25, 1, 3, 5, 7, 11, and 15 days afterdosing, snap-frozen in liquid nitrogen, and stored at�80�C. Priorto analysis, tumor tissues were weighed and homogenized in a3� volume of PBS (containing 40 mg/mL sodium fluoride).Homogenized tissue samples were extracted by protein precipi-tation and analyzed by LC/MS-MS. A Waters SymmetryShieldRP18 column (5 mm, 2.1 � 100 mm) was used with a run timeof 5 minute per sample.

ImmunostainingTumor tissues were harvested from MDA-MB-231 xenograft-

bearingmice 6 hours to 15 days following a single administrationof vehicle, 50 mg/kg irinotecan, or 100 mg/kg STA-12-8666.Samples were fixed in neutral buffered formalin overnight andtransferred to 70% ethanol. Fixed tissues were paraffin-embed-ded, sectioned, and stained by Reveal Biosciences. Briefly, immu-nohistochemical staining for g-H2AX (Cell Signaling Technolo-gies) and hematoxylin counterstaining was performed using aLeica Bond automated immunostainer. A 3DHistech PannoramicSCAN digital scanner (Perkin Elmer) was used for whole bright-field imaging. Quantitative image analysis was performed usingImmunoRatio (18), which calculated the percentage of positivelystained nuclear area for g-H2AX expression (labeling index)through segmentation of diaminobenzidine (DAB)-stained andhematoxylin-stained nuclei regions.

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Mol Cancer Ther; 14(11) November 2015 Molecular Cancer TherapeuticsOF2




ResultsValidation of the HDC mechanism of action

In HDC design, the primary function of the HSP90 inhibitormoiety is to serve as a delivery vehicle. Initial proof-of-conceptstudies utilized an analogous pair of HSP90 inhibitor–attachedfluorescent probes, STA-12-9455 and STA-12-9467 (Supplemen-tary Fig. S1A). Each consisted of a resorcinol-based HSP90 inhib-itor backbone, structurally similar to the small-molecule inhibitorganetespib (19), linked to the dye BODIPY (boron-dipyrro-methene). Replacement of a key hydroxyl with a methoxy inSTA-12-9467 abrogated HSP90 binding and thus this compoundserved as a control for STA-12-9455. BT-20 breast cancer cells weretreated with 5 mmol/L STA-12-9467, STA-12-9455, or BODIPYFLfor 30 minutes, prior to drug wash-out and subsequent imagingover 24 hours (Supplementary Fig. S1B). All three compoundsentered the cells, however, both the naked fluorophore andnonbinding STA-12-9467 displayed only weak cytoplasmic stain-ing after 1 minute and the fluorescent signal for each was lost by30minutes. In stark contrast, strong fluorescence of STA-12-9455was observed for 24 hours, demonstrating that the HSP90-bind-ing conjugate was retained within the cell for this extended timeperiod. Similar effects were seen in multiple tumor lines (unpub-lished observations). Importantly, competitive pretreatment withganetespib was sufficient to prevent intracellular accumulation of

STA-12-9455 (Supplementary Fig. S1C), confirming that com-pound retention was HSP90-dependent.

Evaluation of the chemotherapeutic activity of STA-12-8666, anSN-38–containing HDC

A schematic representation of the structural features of HDC,incorporating an HSP90 inhibitor targeting construct, cleavablelinker, and anticancer payload, is presented in Fig. 1A. For theprototypical cytotoxic payload we selected SN-38, a potent topo-isomerase I inhibitor and metabolite of irinotecan (20). STA-12-8666 comprised a similar resorcinol-based HSP90 inhibitor scaf-fold towhich SN-38was attached via a carbamate linker (Fig. 1B).In this manner, SN-38 is transported in an inactive state untilintracellular cleavage regenerates active chemotherapeutic agentwithin tumors. Enzymatic linker cleavage is primarily achievedthrough carboxylesterase (CES) activity, also involved in prodrugconversion of irinotecan to SN-38 (20). CES are a multigenefamily of serine-dependent esterases involved in the metabolismof endogenous lipids and drugs (21). When assayed in thepresence of purified recombinant CES1b, CES1c, and CES2 pro-teins, STA-12-8666 was similarly cleaved by all three isoenzymesto release SN-38 (Supplementary Fig. S2). As expected, CES2showed the highest catalytic efficiency for irinotecan activation(22). In addition to tumors, CES are present in the blood aswell as

Figure 1.Design, structure, and activity of the SN-38–containing HDC STA-12-8666. A, schematic showing design features of HDC, incorporating an HSP90 inhibitortargeting moiety, cleavable linker, and anticancer payload. B, chemical structure of STA-12-8666. The carbamate linker is shaded in red. C, representative images ofH1975 NSCLC cells treated with 1 mmol/L ganetespib, SN-38, STA-12-8666, or vehicle (DMSO). Cells were exposed to drug for 1 hour; following washout, cellswere cultured for an additional hour prior to staining. Immunofluorescencewasperformed for p-KAP1 induction (red) and cells counterstainedwithDAPI (blue). Scalebar, 200 mm. D, H1975 NSCLC cells were exposed to graded concentrations (10—1,000 nmol/L) of SN-38, STA-12-8666, or ganetespib for 24 hours and celllysates immunoblotted for HSP70, phosphorylated KAP1 (p-KAP1), and g-H2AX as indicated. GAPDH was included as a loading control.

HDC-Mediated SN-38 Drug Delivery

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normal tissues. Moreover, linkages that depend on esterase activ-ity for payload release, such as the one in STA-12-8666, mayalso show species-related differences in activity (23). Therefore,STA-12-8666and irinotecan stabilitywere assessed bymonitoringtheir disappearance in plasma samples from mice, dogs, andhumans (Supplementary Table S1). In mouse plasma, STA-12-8666 levels remained at 81% and 43% of original after 15 and 30minutes, respectively, and were further reduced to 24% by 1hour. Of translational relevance, STA-12-8666 was found to berelatively more stable in human plasma compared to mouse (i.e., 91% vs. 24% at 1 hour). Similar findings were observed foririnotecan (Supplementary Table S1). These data suggest thatSTA-12-8666 may exhibit favorable pharmacology in humans,sufficient to allow tumor accumulation and site-specific pro-cessing by intracellular esterases.

Phosphorylation of KAP1 (KRAB-ZFP–associated protein 1), aprotein involved in DNA repair, has been identified as a readoutfor topoisomerase I inhibition (24). As shown in Fig. 1C, treat-ment of H1975 non-small cell lung cancer (NSCLC) cells witheither SN-38 or STA-12-8666 induced robust p-KAP1 expression.Importantly, HSP90 inhibition alone using ganetespib did not,thus p-KAP1provided a selectivemarker for STA-12-8666payloadactivity. These results were confirmed by immunoblotting (Fig.1D). In addition, g-H2AX is a sensitive indicator of DNA double-strand break formation and additional marker of SN-38 activity(25). Consistent with this, g-H2AX was induced by both SN-38and STA-12-8666 at equivalent concentrations to those thatincreased p-KAP1 levels.

g-H2AX expression can also be indirectly impacted by HSP90inhibition and ganetespib treatment elevated levels of this protein(Fig. 1D). However, ganetespib also more potently upregulatedthe stress-inducible chaperone HSP70, a commonly used bio-marker of HSP90 blockade (26), than STA-12-8666. These datasuggested that, despite robust chaperone binding affinity, theintrinsic HSP90 inhibitory activity of the HDC compound wasrelatively weak. This premise was additionally supported by the invitro antiproliferative activities of the two agents in a panel ofNSCLC cell lines (Table 1).

HSP90 binding is required for HDC activityAs with the fluorophore experiments above, we generated a

corresponding "mock" HDC, STA-12-9432, via modification ofthe 2-hydroxyl group in the resorcinol branch necessary forinteraction with the Asp93 residue within the HSP90 N-terminus(Fig. 2A). Apart from compromised HSP90-binding capacity,STA-12-9432 remained similar to STA-12-8666 in terms of overallstructure and physicochemical profile (Supplementary Table S2).

Molecular characterization of the comparative cellular effects ofSTA-12-8666 and STA-12-9432 were then investigated in vitro

using H1975 cells (Fig. 2B). As expected, dose-dependentincreases in HSP70 expression were observed following STA-12-8666 (albeit at high concentrations) but not STA-12-9432exposure. This differential response was confirmed by HDC-induced destabilization of EGFR (EGFR), the mutant form ofwhich expressed by this line is a highly sensitive HSP90 clientprotein. When p-KAP1 levels were assessed as a surrogate markerfor SN-38 effects, STA-12-8666 and STA-12-9432 promoted com-parable, robust p-KAP1 expression (�300 nmol/L) suggestingthat both compounds underwent effective intracellular cleavageand payload release (Fig. 2B).

Next, SN-38 drug concentrations were measured in H1975xenograft tumors following treatment with either STA-12-8666or STA-12-9432 (Fig. 2C). Initial post-dosing SN-38 levels werecomparable for each compound; however, the degree of accumu-lated SN-38 derived from STA-12-8666 remained relatively con-stant over time, unlike the steady decline observed with STA-12-9432. To functionally discriminate between the HDC and mockcompounds in vivo, the comparative efficacies of irinotecan, STA-12-9432, and STA-12-8666 treatment were evaluated in H1975xenografts (Fig. 2D). Weekly dosing with STA-12-9432 had onlyminimal effects on tumor growth, comparable with irinotecan,consistent with STA-12-9432 acting as a non-HSP90–binding SN-38 prodrug. In contrast, STA-12-8666 treatment completely sup-pressed tumor growth over the course of the study.

Sustained tumor SN-38 exposure following STA-12-8666treatment confers superior growth control to irinotecan

Pharmacokinetic analyses were performed in MDA-MB-231breast cancer xenograft-bearing mice to evaluate payload drugexposures that resulted from STA-12-8666 treatment. Following asingle bolus injection of STA-12-8666 at its highest nonseverelytoxic dose (HNSTD) of 150 mg/kg, HDC levels within the tumorcompartment declined slowlywith time, showing retention out to15 days (Fig. 3A). Confirming expectations, STA-12-8666 treat-ment resulted in extended SN-38 payload release and nanomolarlevels of liberated SN-38 were detectable over this same period(Fig. 3A). The appearance of the cleaved HSP90 inhibitor frag-ment, STA-12-8663, provided further evidence that effectiveHDCcleavage occurred in vivo. This profile was in sharp contrast to thatobserved in irinotecan-treated animals, where comparable tumorconcentrations of SN-38 were initially achieved with a 50 mg/kgdose but levels rapidly declined to belowquantifiable limits by 48hours (Fig. 3B).

In agreement with these data, STA-12-8666 demonstrated long-term growth suppression (�2 weeks) following cessation of drugtreatment in mice bearing MDA-MB-231 tumors, whereas thegrowth inhibitory effects conferred by irinotecan treatment wererapidly lost once drug administration was discontinued (Fig. 3C).When temporal changes in the DNA damage response signaturewere assessed by g-H2AX immunostaining (Fig. 3D), irinotecantreatment promotedonly transient g-H2AXexpression that peaked1 to 3 days following dosing. This contrasted to a more amplifiedand protracted induction that occurred for 10 to 15 days followingSTA-12-8666 exposure. Expression changes in the labeling indexfor each treatment regimen are quantitated in Fig. 3E. A similarkinetic profile was observed when tissues were stained for p-KAP1expression (Supplementary Fig. S3), and administration of theHSP90 inhibitor fragment STA-12-8663 alone to MDA-MB-231xenograft-bearing animals had minimal DNA-damaging effects(Supplementary Fig. S4). Taken together, the strong correlation

Table 1. Comparison of ganetespib and STA-12-8666 in vitro antiproliferativeactivity in NSCLC cell lines

Ganetespib STA-12-8666Cell line IC50 (nmol/L) IC50 (nmol/L)

H292 4 130A549 15 400H1703 17 588H460 18 230H1975 19 242H1299 28 270H358 34 500

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between SN-38 exposure, augmented DNA damage response, anddurable efficacy suggest that topoisomerase inhibition, as a con-sequence of sustained intratumoral release of SN-38, serves as theprimary antitumor mechanism of STA-12-8666.

STA-12-8666exhibits a favorable therapeuticwindowof activityNext, a direct comparison of the therapeutic windows for STA-

12-8666 and irinotecan were undertaken using a clinically rele-vant SCLC model. Mice bearing SR2 xenografts were treated withincreasing doses of both agents on a weekly dosing regimen for 3weeks (Fig. 4A and B). All regimens were well tolerated with nosignificant changes in body weight observed (Supplementary Fig.S5) Irinotecan treatment at its HNSTD of 60 mg/kg resulted indisease stabilization (Fig. 4A). Comparable antitumor activitywith STA-12-8666 was observed at 30 mg/kg, a dose representingapproximately 20% of its HNSTD, and higher doses promotedrobust tumor regressions (Fig. 4B). When efficacy was plotted as afunction of tolerability, i.e., percentage of maximally tolerateddose (MTD) for each agent (67 mg/kg for irinotecan; 200 mg/kg

for STA-12-8666), the curve shift to the right for STA-12-8666underscored a substantially broader window of therapeuticopportunity versus the narrower margin between effective andtoxic dosing seen with irinotecan (Fig. 4C).

HDC exposure is therapeutically superior to combinationirinotecan plus ganetespib treatment

To directly compare STA-12-8666 with combination irinotecanplus HSP90 inhibitor treatment, SR2 tumor-bearing mice wereadministered 4 weekly doses of ganetespib (100 mg/kg) or irino-tecan (50mg/kg), both alone and in combination, or STA-12-8666(150 mg/kg; Fig. 4D). The addition of ganetespib to irinotecanconferred a modest yet nonsignificant improvement in tumorgrowth inhibition over irinotecan monotherapy alone. In starkcontrast, STA-12-8666 was significantly more efficacious thaneither irinotecan or combination therapy and promoted tumorregressions. In termsof tolerability, 3of 8mice diedwhile on-studyin the irinotecan arm, while only one animal was removed fromstudy due to low body weight in the STA-12-8666–treated group.

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Figure 2.HSP90 binding is required for HDC activity. A, chemical structure of STA-12-9432. Modification of the 2-hydroxyl group in the resorcinol branch of the HSP90inhibitor scaffold prevents hydrogen bond formation with Asp93 in HSP90, resulting in loss of chaperone binding activity. B, H1975 NSCLC cells were exposed tograded concentrations (10–1,000 nmol/L) of STA-12-8666 or STA-12-9432 for 24 hours and cell lysates immunoblotted for HSP70, EGFR, and phosphorylatedKAP1 (p-KAP1) expression, as indicated. C, SCID mice bearing established H1975 tumors were treated with 50 mg/kg STA-12-8666 or STA-12-9432 and tumorconcentrations of SN-38 measured 30 minutes and 24 hours after dosing. D, SCID mice bearing H1975 xenografts (n ¼ 7 per group) were intravenouslydosed with vehicle, irinotecan (50 mg/kg), STA-12-9432 (100 mg/kg), or STA-12-8666 (100 mg/kg) on a weekly schedule as indicated. Error bars, � SEM.Animals in the vehicle and irinotecan-treated groups were removed from study when tumors reached 1,500 mm3.






STA-12-8666 is efficacious in tumor models independent ofirinotecan sensitivity

The comparative efficacies of STA-12-8666 and irinotecan ther-apy were then evaluated in models of high and low irinotecansensitivity. As expected, irinotecan treatment induced significant

tumor regressions in the highly sensitive SK-NEP-1 Ewing sarcomaxenograft model; an identical response was achieved with STA-12-8666 (Fig. 5A). Weekly dosing of irinotecan at the same levelexerted minimal antitumor activity against SW780 bladdercancer xenografts, a model that we recently demonstrated is also

Figure 3.STA-12-8666 treatment provides prolonged intratumoral SN-38 exposure and superior growth control to irinotecan. A, SCID mice bearing established MDA-MB-231tumors were administered a single 150 mg/kg dose of STA-12-8666 and tumor concentrations of the intact HDC, HSP90 inhibitor fragment (STA-12-8663), andreleased SN-38 payload were assessed over a 15 day time period. B, mice were treated with a single bolus injection of irinotecan (50 mg/kg) and tumorconcentrations of the prodrug and its metabolite SN-38 were determined for 15 days. C, mice bearing established MDA-MB-231 tumors (n ¼ 8 per group) wereintravenously dosed with vehicle, 50mg/kg irinotecan, or 100 mg/kg STA-12-8666 once weekly for 3 weeks. Tumor growth was monitored for an additional 15 daysfollowing the final drug administration. Error bars, � SEM. (� , P < 0.0001). D, kinetic analysis of g-H2AX induction in MDA-MB-231 xenograft tumors harvested atthe indicated time points ranging from 6 hours to 15 days following a single dose of vehicle, 50 mg/kg irinotecan, or 100 mg/kg STA-12-8666. Representativeimmunostaining patterns for g-H2AX expression from each regimen are presented. Scale bar, 100 mm. E, quantification of g-H2AX labeling index for eachgroup, calculated as the percentage of DAB-stained nuclear area (i.e., g-H2AX positive) over total nuclear area. (STA-12-8666 vs. irinotecan: � , P ¼ 0.0226;�� , P ¼ 0.0013; �� , P ¼ 0.0002; �� , P < 0.0001; ns, not significant).

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insensitive to resorcinol-based HSP90 inhibitors due to endoge-nous glucoronidation (27). STA-12-8666 treatment significantlysuppressed SW780 tumor growth (Fig. 5B), suggesting that con-

jugate stability was less affected by tumor metabolism in thismodel. This was likely due to differential substrate sensitivityand/or expression of UDP-glucuronosyltransferases that are

Figure 4.STA-12-8666 has a broad therapeutic window and is more efficacious than combination irinotecan plus HSP90 inhibitor treatment. A, SCID mice bearingestablished SR2 tumors (n ¼ 5 mice/group) were intravenously administered graded concentrations of irinotecan on a weekly dosing regimen, asindicated. Data are expressed as mean tumor volumes � SEM. B, mice bearing established SR2 tumors were intravenously administered gradedconcentrations of STA-12-8666 on a weekly dosing regimen. C, the relationship between dosing and activity for each agent presented in A and B. Tolerabilityis plotted as a percentage of the respective maximally tolerated doses (MTD) for each drug, and efficacy measured as either percentage tumor growthinhibition (T/C values) or regression seen 8 days following the final dose. D, SR2 tumor-bearing mice (n ¼ 8 per group) were dosed weekly with vehicle,ganetespib (100 mg/kg), irinotecan (50 mg/kg), the combination of irinotecan plus ganetespib, or STA-12-8666 (150 mg/kg) as indicated. Data are expressedas mean tumor volumes � SEM. (� , P < 0.0001; ns, not significant).

Figure 5.STA-12-8666 has potent therapeuticactivity independent of irinotecansensitivity. A, mice bearing SK-NEP-1Ewing sarcoma xenografts (n ¼ 6 pergroup) were dosed with vehicle,irinotecan (50mg/kg), or STA-12-8666(150 mg/kg) on a weekly schedule asindicated (ns, not significant). B, SCIDmice bearing SW780 bladder cancerxenografts (n ¼ 8 per group) weredosed weekly with vehicle, irinotecan(50 mg/kg), ganetespib (100 mg/kg),or STA-12-8666 (150 mg/kg) asindicated. (�, P < 0.0001).






responsible for glucuronidation in these cells. Furthermore, treat-ment of SW780 cells in culture with ganetespib resulted in com-pletemetabolismof theHSP90 inhibitorwithin 24hours,whereassignificant levels of intact STA-12-8666 (or a nonresorcinol inhib-itor control) remained present in the media over the same timeperiod (unpublished observations).

STA-12-8666 treatment induces durable regressions andcontrols tumor growth with intermittent dosing in aggressivetumor models

Extended xenograft modeling of aggressive human disease wasused to evaluate the durability of response to STA-12-8666 (Fig.6A). Mice bearing pancreatic cancer PDX were administered 3once-weekly doses of vehicle, STA-12-8666, or irinotecan andtumor growthmonitored out to 150 days. Vehicle and irinotecan-treated animals quickly progressed and were removed from studyafter 1 month. Complete regressions occurred in all five animalsbearing nine tumors that were administered STA-12-8666; aresponse that was maintained for at least 2 months followingcessation of initial therapy. At day 110, the first two animals topresent with four recurring tumors were retreated with weeklySTA-12-8666 dosing. All four tumors responded with furtherregressions and no regrowth was observed. Similarly, two addi-tional mice with three recurrent tumors were administered asecond round of STA-12-8666 at day 124 and tumor growth wascompletely suppressed. The remaining animal did not receive asecondary cycle of STA-12-8666 and its two tumors showedregrowth 3 months after initial treatment, underscoring the dura-ble nature of disease control. Importantly, all recurrent tumorsretained full sensitivity to subsequent therapeutic HDC challenge,suggesting that acquired resistance to STA-12-8666 was notresponsible for regrowth.

Finally, intermittent drug dosing regimens were challengedexperimentally by allowing H1975 xenografts to propagate tolarge volumes (�750 mm3) prior to treatment. Mice were dosedwith vehicle, irinotecan once a week, or STA-12-8666 on a weekly

or biweekly schedule (Fig. 6B). Vehicle-treated tumors doubled involume within 5 days, accentuating the high proliferative poten-tial of these large neoplasms. Weekly irinotecan therapy exertedminimal growth suppressive effects, in marked contrast to theimmediate tumor stabilization anddramatic regressions observedfollowing STA-12-8666 exposure on the same schedule. Of note,the less frequent, 1�/2 week STA-12-8666 dosing regimen wassimilarly efficacious and induced significant tumor regressionsover the course of the study (Fig. 6B).

DiscussionSTA-12-8666 is an HDC carrying SN-38, prototypical of a new

class of therapeutic agents designed to provide selective drugaccumulation and durable chemotherapeutic exposures withintumors. Aunique aspect ofHDC technology is that it employs onesmall-molecule entity (i.e., HSP90 inhibitor) as a targetingmoietyfor tumor-directed delivery of another that serves as a cytotoxicpayload. Preferential tumor retention is characteristic of small-molecule inhibitors of HSP90 and, while the underlying basis ofthis effect remains to be fully elucidated (14, 15), this feature isalso currently being exploited in the development of new func-tional imaging and therapeutic monitoring approaches (28–30).Here we report that the HSP90-binding capacity of STA-12-8666was both necessary and sufficient to promote intratumoral local-ization and retention of the HDC molecule, resulting in pro-longed SN-38 exposure and optimized therapeutic indices for thetopoisomerase I inhibitor. While we cannot rule out a contribu-tion of direct chaperone inhibitory effects by STA-12-8666, thesewere unlikely exerting a major influence as evidenced by thecompounds' weak intrinsic HSP90 inhibitory activity, as well asthe superior efficacy achieved when compared with irinotecanplus ganetespib combination therapy. Further evidence was pro-vided by the comparativelyweaker antiproliferative effects of STA-12-8666 on tumor lines in vitro relative to ganetespib, yet therobust tumor regressions seen in xenograft lung tumors in vivo

Figure 6.STA-12-8666 induces durable tumor regressions in aggressive xenograft models. A, pancreatic cancer PDXs were established in SCID mice and animalsadministered an initial 3-week cycle of either irinotecan (50 mg/kg) or STA-12-8666 (150 mg/kg). Irinotecan-treated mice (average tumor volumes presented)rapidly progressed and were removed from study after 1 month. The first 2 HDC-treated mice to progress were administered a second 5-week cycle ofSTA-12-8666 beginning at day 110; the next 2 animals with recurrent tumors were re-treated on days 124 and 131. All recurrent PDX tumors to receive a secondround of STA-12-8666 dosing underwent further, complete regressions. B, H1975 xenografts were allowed to grow to an average volume �750 mm3 prior toinitiation of drug treatment. Dosing on either a weekly (days 0, 7, 14, 21, 28) or every other week schedule (days 0, 14, 28) with STA-12-8666 inducedsignificant tumor regressions.

Proia et al.





exceeded ganetespibs' previously published antitumor activity inthe samemodels (31, 32). Thus, our findings clearly demonstratethat HDC-mediated delivery of an established cytotoxic agent canachieve dramatic improvements in efficacy over traditional clin-ical formulations for these agents and that the approach is notcomplicated by effects due to loss of HSP90 function. Interest-ingly, even modest, nonproteotoxic effects on HSP90 inhibitionhave been shown to limit the emergence of resistance to antican-cer chemotherapeutics (33); in this regard, STA-12-8666 mayretain an inherent capacity to augment SN-38 activity via restrict-ing potential development of topoisomerase inhibitor resistance.Overall, the HSP90 inhibitor backbone in STA-12-8666 func-tioned as an effective targeting constructwith theprincipal cellularimpacts arising from sustained SN-38 release.

SN-38 was chosen as an appropriate payload due to its broadclinical potential, well-established pharmacologic profile, andrelative ease of synthetic incorporation into the HDC construct.A number of issues, including restricted active drug bioavailabilityand poor solubility (34, 35), have stimulated considerable inter-est in utilizing irinotecan and/or SN-38 in alternate deliveryformulations intended to improve drug pharmacokinetics andtherapeutic activity. Such investigational agents include the ADCsIMMU-132 and epratuzumab-SN-38 (36–38); polymer–drugconjugates (PDC) EZN-2208 (39) and etirinotecan pegol (40);and polymer-based nanoparticle therapeutics like NK012 (41). Anumber of unique characteristics differentiate the HDCmodalityfrom these approaches. For example, unlike ADCs, HDCs do notrequire recognition of unique cell surface antigens (typically withrestricted tumor distributions) and subsequent endocytosis forcellular uptake. Instead, tumor-expressedHSP90 provides a ubiq-uitous intracellular "sink" to attract the conjugate compound and,as small molecules, cellular entry and accumulation is achievedthrough either passive diffusion or common active transportmechanisms. In addition, highly potent "ultratoxic" chemother-apeutic payloads are typically required to achieve sufficient ADCantitumor activity (42) and HDC construction is not restricted bysuch constraints. Furthermore, nanoparticle and PDC macromo-lecular therapeutics are acutely reliant on the enhanced perme-ation retention effect (EPR) for passive accumulation into solidtumors (43) and optimal benefit is often mitigated by a variety ofphysiologic obstacles (44).HDC-based therapy thus represents analternative strategy to provide site-specific accumulation anddurable exposure of chemotherapeutic agents to tumors but withwider applicability and without existing limitations of ADC orPDC delivery.

Confirming expectations, STA-12-8666 showed a favorablebiodistribution and activity profile, with tumor-selective accumu-lation of the compound acting as a reservoir for sustained intra-cellular cleavage and SN-38 payload release. This was validated bypersistent drug activity and DNA damage following STA-12-8666dosing, in contrast to the more restricted kinetics obtained withirinotecan treatment. This characteristic was directly related to thedegree of tumor growth control exhibited by STA-12-8666;accordingly, the extended tumor residency periods and mainte-nance of SN-38 at therapeutic levels promoted robust anddurableantitumor activity in vivo. Indeed, STA-12-8666 demonstratedremarkable efficacy in suppressing aggressive xenograft tumorgrowth and promoting durable regressions across a variety ofcancer types, including tumors refractory to irinotecan treatment.Irinotecan is indicated for use inmetastatic colorectal cancer (16),and adjuvant activity has been observed in a range of other

malignancies (45). This profile suggests quite broad-spectrumclinical activity for the topoisomerase inhibitor; in practical terms,however, a number of factors have prevented widespread use ofthis agent, including disappointing response rates, transient clin-ical responses, major dose-limiting toxicities, and drug resistance(46). Importantly, the antitumor effects of topoisomerase I inhi-bition appear more proportionate to length of drug exposurerather than the concentration of the inhibitor used (47). Thus, thecontrolled release of cytocidal levels of SN-38 within the tumorcompartment for an extended period following HDC adminis-tration was sufficient to account for the superior antitumoractivity and broader therapeutic window achieved with STA-12-8666 exposure comparedwith irinotecan therapy. In light of theseconsiderations, it is reasonable to suggest that HDC-mediateddelivery represents an innovative strategy for extending the clin-ical applicability of this type of agent into additional indicationswhich, due to either insufficient drug exposures and/or narrowsafety margins, were previously not considered possible and thefeasibility of this approach warrants further evaluation.

Two further considerations with direct translational relevanceemerged from this study. First, robust tumor regressions inNSCLCxenograft tumors permitted to grow to large volumes beforetreatment was initiated were achieved with infrequent dosing ofSTA-12-8666, a finding that not only underscores a profoundresponse to HDC therapy but also supports the potential use ofintermittent dosing schedules within the clinical setting. Second,in a highly aggressive human pancreatic PDX model, STA-12-8666 therapy significantly outperformed irinotecan to inducedurable tumor regressions following one cycle of treatment. Moreimportantly, recurrent tumors remained sensitive to subsequenttherapeutic challenge with STA-12-8666, indicating that tumorregrowth was not due to the emergence of selected clones withacquired resistance to the HDC compound. It is reasonable tosuggest, therefore, that HDC delivery of SN-38 may represent aneffectivemeans to circumvent commonmechanisms of resistancethat frequently develop in response to irinotecan therapy (16).

In summary, we have developed and characterized a uniqueHDC agent that employs a distinct mechanism of targeted drugdelivery to achieve potent and sustained antitumor effects. STA-12-8666 displays optimal pharmacologic properties, includinghigh tumor retention and controlled cytotoxic payload release,which combine to produce a remarkable therapeutic index. Thesefindings thus identify STA-12-8666 as a promising new therapeu-tic candidate for cancer treatment.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interests were disclosed.

Authors' ContributionsConception and design:D.A. Proia, N. Tatsuta, S. Osman, D. Chimmanamada,W. YingDevelopment of methodology:D.A. Proia, D.L. Smith, J. Zhang, J. Sang, S. He,T. Inoue, N. Tatsuta, S. Osman, W. YingAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D.A. Proia, D.L. Smith, J. Zhang, J.-P. Jimenez,J. Sang, L.S. Ogawa, M. Sequeira, J. Acquaviva, S. He, C. Zhang, V. Khazak,I. Astsaturov, S. Osman, W. YingAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D.A. Proia, J. Zhang, J. Acquaviva, S. He, V. Khazak,I. Astsaturov, N. Tatsuta, S. Osman, R.C. Bates, D. Chimmanamada, W. YingWriting, review, and/or revision of the manuscript: D.A. Proia, I. Astsaturov,N. Tatsuta, S. Osman, R.C. Bates, W. Ying






Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D.A. Proia, D.L. Smith, J. Zhang, S. He,V. Khazak, S. Osman, D. Chimmanamada, W. YingStudy supervision: D.A. Proia, D.L. Smith, J. Sang, T. Inoue, N. Tatsuta,D. Chimmanamada, W. Ying

AcknowledgmentsThe authors thank themanymembers of the Synta team for contributions in

STA-12-8666 design and development, and helpful discussions pertaining tothis work. In particular, the authors thank Pat Rao and John Chu for performing

the affinity binding assays. In addition, the authors recognize their collaboratorsat Fox Chase Cancer Center for their helpful suggestions on the manuscript,Natalya Skobeleva for her work on the PDXmodels, and Reveal Biosciences forimmunohistochemistry.

Grant SupportThis work was funded by Synta Pharmaceuticals Corp.

Received June 1, 2015; revised August 7, 2015; accepted August 7, 2015;published OnlineFirst August 13, 2015.

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Published OnlineFirst August 13, 2015.Mol Cancer Ther David A. Proia, Donald L. Smith, Junyi Zhang, et al. Delivery of Topoisomerase I Inhibitor to Tumors

SN-38 Conjugate Strategy for Targeted−HSP90 Inhibitor

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