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Cancer Therapy: Clinical

Tumor Survivin Is Downregulated by the Antisense OligonucleotideLY2181308: A Proof-of-Concept, First-in-Human Dose Study

Denis C. Talbot1, Malcolm Ranson2, Joanna Davies1, Michael Lahn3, Sophie Callies4, Val�erie Andr�e4,Sunil Kadam3, Michael Burgess4, Christopher Slapak3, Anna L Olsen1, Peter J. McHugh1,Johann S. de Bono5, Julian Matthews6, Azeem Saleem6, and Patricia Price7

AbstractPurpose: Enhanced tumor cell survival through expression of inhibitors of apoptosis (IAP) is a hallmark

of cancer. Survivin, an IAP absent from most normal tissues, is overexpressed in many malignancies and

associated with a poorer prognosis. We report the first-in-human dose study of LY2181308, a second-

generation antisense oligonucleotide (ASO) directed against survivin mRNA.

Patients and Methods: A dose-escalation study evaluating the safety, pharmacokinetics, and pharma-

codynamics of LY2181308 administered intravenously for 3 hours as a loading dose on 3 consecutive days

and followed by weekly maintenance doses. Patients were eligible after signing informed consent, had

exhausted approved anticancer therapies and agreed to undergo pre- and posttreatment tumor biopsies to

evaluate reduction of survivin protein and gene expression.

Results: A total of 40 patients were treated with LY2181308 at doses of 100 to 1,000 mg. Twenty-six

patients were evaluated at the recommended phase 2 dose of 750 mg, at which level serial tumor sampling

and [11C]LY2183108 PET (positron emission tomography) imaging demonstrated that ASO accumulated

within tumor tissue, reduced survivin gene and protein expression by 20% and restored apoptotic signaling

in tumor cells in vivo. Pharmacokinetics were consistent with preclinical modeling, exhibiting rapid tissue

distribution, and terminal half-life of 31 days.

Conclusions: The tumor-specific, molecularly targeted effects demonstrated by this ASO in

man underpin confirmatory studies evaluating its therapeutic efficacy in cancer. Clin Cancer Res; 16(24);

6150–8. �2010 AACR.

Survivin, a 16.5-kDa protein encoded by the essentialgene BIRC5, was originally identified as an inhibitor ofapoptosis (IAP) that exerts its effects through binding toSMAC (second mitochondrial activator of caspases) pre-venting the sequestrationof IAPsby SMACand inhibitionofcaspase-dependent apoptosis (1). As a component of thekinetochore-associated complex, survivin also plays animportant role in the regulation of latemitosis and cytokin-esis (2). Survivin is expressed in a wide range of humancancers andwhen overexpressed, is associatedwith a poorerprognosis (3). With the exception of placenta, thymus,activated T cells, gastrointestinal crypt cells, and regenerat-ing liver, survivin is not expressed in normal adult tissue (4).Thus, survivin represents an attractive molecular target fortherapeutic intervention. Targeted approaches against sur-vivin include small molecule inhibitors against the survivinprotein, gene silencing, and survivinmRNAblockade (5, 6).The 20-O-methoxyethyl–modifiedASO(secondgeneration)LY2181308 is an 18-mer ASO that binds to the translationinitiation codon of the survivin transcript. Following ASOhybridization to survivinmRNA, RNase H–dependent clea-vage of the duplex ensues with subsequent degradation ofthe survivin mRNA (7). Thus, survivin protein expression is

Authors' Affiliations: 1Department of Medical Oncology, University ofOxford, Oxford Radcliffe Hospitals NHS Trust, Oxford, 2School of Cancerand Enabling Sciences, MAHSC, University of Manchester, Christie Hos-pital NHS Foundation Trust, Manchester, United Kingdom; 3Early Oncol-ogy Clinical Investigation, Eli Lilly & Co, Indianapolis, Indiana; and 4Eli Lilly& Co, Erl Wood Research Centre, Windlesham, 5Institute of Drug Devel-opment, Royal Marsden, Sutton, 6Wolfson Molecular Imaging Centre,MAHSC, University of Manchester, and 7Academic Radiation Oncology,The Christie Hospital, National Health Services (NHS) Foundation Trust,University of Manchester, Manchester, United Kingdom

List of previous presentations related to this article: D.C. Talbot,J. Davies, S. Callies, V. Andre, M. Lahn, J. Ang, J. De Bono, M. Ranson.First human dose study evaluating safety and pharmacokinetics ofLY2181308, an antisense oligonucleotide designed to inhibit survivin.ASCO Abstract 3518, Oral Presentation, Developmental Therapeutics:Molecular Therapeutics, June 1, 2008.

D.C. Talbot, J. Davies, A.L. Olsen, V. Andr�e, M. Lahn, E. Powell, S. Kadam,J. De Bono, P.J. McHugh, M. Ranson. Pharmacodynamic evaluation ofLY2181308 in Patients with metastatic malignancies. Abstract 3507, OralPresentation, Clinical Science Symposium, May 30, ASCO 2009.

Corresponding Author:DenisC. Talbot,Department ofMedicalOncology,University of Oxford, Cancer and Haematology Center, Churchill Hospital,OldRoad,Headington,OxfordOX37LJ,UnitedKingdom.Phone:44 (0)1865235312; Fax: 44 (0)1865 235985; E-mail: [email protected].

doi: 10.1158/1078-0432.CCR-10-1932

�2010 American Association for Cancer Research.

ClinicalCancer

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specifically inhibited without affecting expression of othergenes including other IAP proteins (8). Compared withphosphorothioate or first-generation ASOs, second-genera-tion ASOs are more stable, have an improved pharmaco-kinetic (PK) profile, increased potency, and reduced toxicity(9). Using aggregate data from preclinical pharmacologyand toxicology, an integrated PK/PD (pharmacodynamic)model was developed to predict a biologically effective doserange for clinical evaluation (10).On thebasis of this, a first-in-human dose (FHD) study was designed to evaluate thebiodistribution profile of LY2181308 by measuring thefollowing tumor-specific PD changes: tumor tissue penetra-tionof LY2181308, downregulationof tumor survivin at themRNA, and protein levels and restoration of tumor apop-tosis at a dose and schedule that was safe in humans. Tothis end, pre- and posttreatment tumor sampling was per-formed for rigorous assessment of target modulation (i.e.,survivin protein and gene expression), the effect of restoringnormal apoptosis/cell cycle–related protein activity andbiodistribution of LY2181308 including subcellular locali-zation.

Patients and Methods

Study designThis FHD monotherapy study was divided into 3 parts:

part A (safety, PK), 1-patient cohorts with initial 100 mgand escalated, by dose doubling, to 400 mg; part B (safety,PK, PD), 3-patient cohorts with 50% dose escalation untildose-limiting toxicity (DLT); part C (dose confirmationcohort, PK, PD). The transition from part A to part B wasplanned on the basis of either development of toxicity orevidence from PK that the predicted biological effectivedose (BED) had been reached. The recommended dose forphase 2 studies was determined from both DLT and the

reduction in survivin expression in tumor tissue at theanticipated BED as defined by the preclinical PK/PDmodelof Callies et al. (10). At the recommended phase 2 dose, 2companion studies were conducted at the Universities ofOxford (study 1, endobronchial tumor sampling) andManchester (study 2, [11C]LY2181308 study). The entirestudy was approved by the Medicines and Healthcareproducts Regulatory Agency (MHRA) and a MulticentreResearch Ethics Committee (MREC).

Enrollment criteriaInclusion criteria were as follows: at least 18 years of age,

confirmedmalignancy, exhausted approved standard thera-pies, tumor accessible for biopsy, written informed consent,Eastern Cooperative Oncology Group (ECOG) perfor-mance status of 0 or 1, discontinued previous anticancertherapies, absolute neutrophil count � 1.5 � 109/L, plate-lets � 100 � 109/L, hemoglobin � 9 g/dL, normal serumbilirubin <2.5� upper limit of normal, alanine transami-nase (ALT) and aspartate transaminase (AST), calculatedcreatinine clearance � 50 mL/min, normal activated pro-thrombin time (aPTT), and prothrombin time (PT), andcontraceptive precautions taken. Exclusion criteria were asfollows: bleeding diathesis, major surgery within last 4weeks, pregnant or lactating, symptomatic central nervoussystem (CNS) neoplasm, taking concomitant anticoagulanttherapy, received prior ASO, treatment with an unapproveddrug, positive test results for viral infection.

Drug formulation and schedule of administrationLY2181308 was diluted in 500 mL of normal saline for

intravenous injection and infused as a 3-hour infusion on 3consecutive days as a loading dose, followed by weeklymaintenance doses. The dose and dose schedule were basedon preclinical safety, PK and pharmacology studies ofLY2181308. These were integrated within a PK/PD modelthat predicted the BED (10).

Treatment assessmentDLT was defined (CTCAE Version 3.0; ref. 11) as 1 of 3

patientswith the following: grade3/4hematologic toxicitiesmore than 5 days, aPTT prolongation more than 48 hoursafter infusion, any grade 3/4nonhematologic toxicity or anyother DLT including those associated with sustained com-plement activation. Laboratory examinations: C-reactiveprotein (CRP), complement split products (National JewishMedical and Research Center), hematology, serum chem-istry. Pretreatment tumor biopsy: after enrollment andbefore first loading. Posttreatment tumor biopsy: windowof 48 to 96 hours after the third loading dose. Descriptivestatistics were used for all patients receiving at least 1 dose ofLY2181308 to evaluate safety, PK, and efficacy.

Pharmacokinetic assessmentPK parameters were analyzed (WinNonlin Enterprise,

Version 5.2) for: time of maximum concentration (Tmax),maximum plasma concentration (Cmax), area underthe plasma concentration versus time curve (AUC), and

Translational Relevance

Evasion of apoptosis is a hallmark of malignancy.Survivin is a small, naturally occurring inhibitor ofapoptosis (IAP) that is often highly expressed by cancercells making it an attractive target for therapeuticintervention. The antisense oligonucleotide (ASO)LY2181308 downregulates survivin by targeting thesurvivin transcript and has antitumor efficacy throughinduction of tumor cell apoptosis in preclinical models.This First-In-Human (FHD) study demonstrates theproof of concept that ASO therapy directed againstsurvivin mRNA reduces survivin mRNA and proteinlevels and restores apoptosis in tumors of cancerpatients. The research represents a critical step in thetranslation from discovery of survivin, an importanttumor-related IAP, to successful clinical application ofsurvivin-targeted therapy. Finally, this FHD study helpsto better understand how ASOs can be used as novelagents in the treatment of cancer.

Tumor Survivin Is Downregulated by LY2181308

www.aacrjournals.org Clin Cancer Res; 16(24) December 15, 2010 6151




clearance (CL) following the third and fifth administrationof LY2181308 on days 3 and 15. Plasma PK data werepooled for nonlinearmixed affectmodeling analysis (NON-MEM, Version 6.1) to determine the compartmental PKparameters: mean value, variance, and subject variability.The interindividual variability was coded as an exponentialmodel and the residual variability as a proportional model.

Development of a specific antibody to detect the ASOA polyclonal rabbit antibody against the ASO was gen-

eratedusing a keyhole limpet hemocyanin (KLH)–modifiedASO (Lampire Biological Laboratories). It was validated foruse in formalin-fixed, paraffin-embedded specimen utiliz-ing Ventana’s DiscoveryXTstaining platforms (Ventana).

Pharmacodynamic assessmentFormalin-fixed tissue was used for immunohistochem-

ical assessment at Ventana (Tucson) to determine: ASO,survivin (NB500-201; Novus Biologicals), cleaved caspase3 (CC3; CST9661; Cell Signaling Technologies), and Ki67(mouse monoclonal antibody; Ventana). All antibodieswere detected by appropriate secondary antibodies (VectorLaboratories). Staining intensity was determined using theHSCORE and Ventana’s automated imaging software (12).Gene expression analysis (Panomics; ref. 13) was used toquantify Survivin mRNA expression (branched DNA,bDNA). Percentage change in protein and mRNA expres-sion was summarized using medians and approximately95% confidence limits (14).

Endobronchial biopsyFiberoptic endobronchial tumor sampling is a safe,

short, and standard procedure (15). Endobronchial tumorswere visualized and sampled by brushing with a 1-mmbrush. The samples were processed immediately for flowcytometry assessment.

Flow cytometryBriefly, disaggregated tumor cells were fixed in formal-

dehyde and subsequently stained with phycoerytherin-(PE)conjugated monoclonal antibodies directed againstsurvivin (Clone 91630; R&D Systems). Flow cytometricdata (obtained on a CyAN machine, Dako) were analyzedusing Summit software (Dako).

[11C]LY2181308 PET imagingAfter establishing safety in nonhuman primates (16),

patients received less than 1 mg and less than 600 MBq of[11C]LY2181308 prior to LY2181308, and during themaintenance infusion on day 15 (17). PET data werecollected for 90 minutes following bolus radiotracerinjection.

FDG-PET imagingFDG-PET imaging was performed prior to treatment with

LY2181308 and on day 22. The imaging was performed aspreviously recommended (18) and consistent with institu-tional radiation guidelines.

Results

Between October 2004 and December 2008, 40 patientswere enrolled in this monotherapy FHD study, of whom 17were enrolled in the initial dose-escalation stage with dosesranging from 100mg to 1,000mg. Twenty-six patients weretreated at the 750 mg dose level, including 6 patients in 2site-specific studies: study 1 assessed apoptosis and cell-cycle progression changes by flow cytometry of endobron-chial tumor cells in 3 patients with NSCLC (non–small celllung carcinoma); study 2 examined the biodistribution of[11C]LY2181308measured by PET in 3 patients. The demo-graphics of the study population was typical of phase 1oncology trials (19) with the majority of patients havingreceived prior chemotherapy (Table 1). Fourteen patientswith hepatic metastasis were included, of whom 11 hadbiopsies taken from liver lesions. LY2181308 was welltolerated with the majority of the patients showing grade1 or 2 toxicities (29/40, 72.5%). Of the 40 patients, 11patients (27.5%) had grade 3 or 4 toxicities (Table 2). Twoof the 11 patients received the 1,000 mg dose. One patienthad a grade 3 hypophosphatemia, whereas the otherpatient experienced a grade 4 lymphopenia and a pro-longed Grade 3 headache, which did not respond to painmedication. This event was defined as the DLT for thisstudy. Concurrently, a sharp rise in CRP was observed inthis patient raising the concern of a possible complement-

Table 1. Baseline patient and disease charac-teristics (n ¼ 40)

Sex n (%)Male 19 (47.5)Female 21 (52.5)

Age group<65 32 (80.0)>65 8 (20.0)

ECOG performance status0 15 (37.5)1 25 (62.5)

Pathologic diagnosis (n)Gastrointestinal tumors

(including 7 colon,1 rectal, 2 gastric,1 esophageal,1 pancreas cancer)

12 (30.0)

Breast cancer 8 (20.0)Melanoma 7 (17.5)Lung cancer 7 (17.5)Other (3 sarcoma,

1 ovary, 1 headand neck, 1 unknownadenocarcinoma)

6 (15.0)

Prior therapyRadiotherapy 23 (57.5)Surgery 30 (75.0)Chemotherapy 39 (97.5)

Talbot et al.

Clin Cancer Res; 16(24) December 15, 2010 Clinical Cancer Research6152




induced cerebrospinal leak syndrome. Three patients weretreated at the 900 mg dose level and no DLTs wereobserved, but CRP, AST/ALT, and moderate flu-like symp-toms occured during the loading doses, suggesting con-tinued complement-activation. The most commonsymptoms were flu-like syndrome (fever, rigor, musculos-keletal pain, nausea), fatigue, and vomiting (Table 2). Themost frequent laboratory toxicity was prolongation of aPTT(generally grade 1), which was observed at the time of theinfusion in 75% of the patients. Other laboratory abnorm-alities, not considered as severe adverse events by investi-gators, included lymphopenia (70%), thrombocytopenia(38%), hypokalemia (38%), and anemia (35%). Transientincreases in complement Bb were noted at the end of thethird loading dose, but was not associated with adverseclinical events. Given the comparable PK profile of thehigher doses of 900 and 1,000 mg to the 750-mg dose, therecommended dose of LY2181308 for further clinical stu-dies using the same dosing schedule was defined as 750mg,a dose likely to be safe in combination with chemother-apeutics.

The PK properties of LY2181308 and reduction ofsurvivin expression in tumor tissue were assessed bycollecting plasma samples and tumor biopsies beforeand after completing the loading dose. In parts B andC of the protocol, 31 patients were enrolled with 26(84%) agreeing to have both pre- and posttreatmentbiopsies. Ten patients had either insufficient tumor mate-rial in the posttreatment biopsy or endogenous pigments(bilirubin, hemosiderin, and melanin) that interferedwith IHC staining. Hence, in 16 (52%) patients, weobtained sufficient pre- and postdosing tumor tissue toassess the PD effects of LY2181308 by protocol-definedlaboratory procedures (Fig. 1A–F).

Survivin protein expression as measured by IHC wasreduced by 21%, concomitant with a statistically significant20% reduction inmRNA expression (P < 0.05) as shown bygene expression analysis (Fig. 1G). Because of the highintensity of nuclear survivin IHC staining prior to thetreatment, reduction of nuclear protein expression wasmore clearly detected than changes seen in the cytoplasm(Fig. 1E–G). Similarly, in the site-specific study inNSCLC, a

Table 2. Study drug related adverse events occurring in 2 or more patients (n ¼ 40)

CTCAE description Maximum CTC grade

1 2 3 4

LaboratoryPartial thromboplastin time 28 2Platelet counts 7 2ALT/SGPT (serum glutamic pyruvic transaminase) 5 2Metabolic/laboratory—other (including CRP) 5 1Hemoglobin 3 2Lymphopenia 2 1 1 1AST/SGOT (serum glutamic oxaloacetic transaminase) 3 1Phosphate (hypophosphatemia) 1 3Potassium (hypokalemia) 4Glucose (hyperglycemia) 2 1Leukocytes (total WBC) 3Blood/bone marrow—other (eosinophils, etc) 2 1Alkaline phosphatase 1 1Bilirubin (hyperbilirubinemia) 2Sodium (hyponatremia) 1 1

NonlaboratoryFever (in the absence of neutropenia) 9 4 1Nausea 7 3 1Fatigue (asthenia, lethargy, malaise) 6 3 1Vomiting 6 1Diarrhea 2 1Pain neurology—head/headache 2 1Rigors/chills 2 1Flu-like syndrome 1 1Hypotension 1 1Pain musculoskeletal-–joint 2Sweating (diaphoresis) 2

Abbreviation: CTC, circulating tumor cells.






reduction of survivin protein expression was demonstratedby flow cytometry in endobronchial tumor cells comparedwith pretreatment levels in all 3 patients studied (Fig. 1I).

The PK of LY2181308 confirmed a multiphasic dispo-sition in plasma with rapid tissue distribution and half-lives of 0.5, 2.5, and 12 hours, and an elimination half-life of 31 days (Fig. 2A). The terminal half-life was theresult of a clearance of 20 to 30 L/h and volume ofdistribution of more than 1,000 L consistent with thetissue distribution of LY2181308 of approximately 90%.As described for other ASOs (9), the primary sites ofnormal tissue uptake of LY2181308 were renal and hepa-tic, with less uptake seen in other normal tissue (17). TheASO was detected by IHC in tumor tissue in 10 of 11patients, either within tumor cells or tumor-associatedmacrophages (Fig. 2B and C) and in 5 of 11 patients instroma (Fig. 2D and E). Compatible with the IHC stain-ing, the [11C]LY2181308 PET imaging confirmed that

following administration of 1 mg of [11C]LY2181308,tumor penetration of LY2181308 ranged from 21 to 84 ngh/mL tumor with a maximum concentration that rangedfrom 10 to 60 ng/mL. These levels are comparable withthe concentration measured by ELISA in tumor biopsyand with that required for target inhibition (Table 3). ThePK profile and tissue distribution of LY2181308 wereconsistent with the PD effects of the agent describedabove. However, there was no evident relationshipbetween the localization of the ASO within the tumortissue compartments, clinical response, or change insurvivin expression, perhaps due to the limited numbersof patients included within each cohort.

Of 22 patients assessed for efficacy using the RECIST(response evaluation criteria in solid tumors) criteria (20),a total of 4 patients achieved stable disease, including 1patient with metastatic melanoma who remained free ofdisease progression for 18 months. Reduction of survivin

10o 101 102 103 104

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Fig. 1. Reduction of survivin protein and mRNA expression after loading dose of LY2181308. IHC of tumor biopsy from 1 representative patient with breastcancer (A–F) is shown prior to (A, C, E) and following (B, D, F) LY2181308 treatment stained with H&E (1� in A and B; 20� in C and D) and survivin antibody (20�in E, F). G, percent change of survivin mRNA and protein (nuclear and cytoplasmic) expression by IHC with medians and 95% confidence limits. Flowcytometric analysis of survivin expression by endobronchial NSCLC obtained from 3 patients by fiberoptic-guided bronchial brushing prior to and afterLY2181308 (H) and change in high survivin-expressing cells (I). PE, phycoerythrin.

Talbot et al.





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Fig. 2. Plasma and tissue PKs following 750-mg dose of LY2181308. Plasma PK following loading and maintenance dosing (A). Observed values(open circles) are depicted with predicted exposures (median 5th and 95th percentiles). Tissue levels of LY2181308 detected by IHC in 2 representativepatients (B–E) before (B, D) and following loading dose (C, E) in tumor, 10 of the 11 patients showing a similar pattern (B, C), and in stromal cells, 5 of the11 patients showing a similar pattern (D, E). Tissue PKs assessed by [11C]LY2181308 uptake (AUC during 90 minutes scan) scaled between the 0 and140 ng h/mL window for a 1-mg dose: prior to first loading dose (F); and during the second half of the maintenance dose on day 15 (G). Inserts highlight changein uptake in specific area of mesothelioma over time.

Table 3. Plasma and tissue PKs of LY2181308 at 750 mg

Predicted1,000 simulations

Observedn ¼ 24

Plasma PKAUC0–24, ng h/mL 283,725 342,794

(133,922–582,041) (187,344–603,944)Cmax, ng/mL 65,489 69,120

(42,838–96,897) (39923–155,514)Cmin, ng/mL 93.8 73.8

(63.9–131) (36.8–135.9)

1,000 simulations 11C-PET ELISA(n ¼ 4) (n ¼ 5)

Tumor tissue PKsConcentration, ng/mL 33.2 32.5 22.4

(18.8–54.0) (13.9–52.8) (3.64–87.4)

NOTE: Values given are mean (range).






expression was associated with an increase in the apoptosismarker CC3 and a reduction in tumor expression of theproliferation marker Ki67 (Fig. 3A and B). In the singlepatient assessed, a partial metabolic response in severalmesothelioma lesions was observed with a 40% reductionin standard uptake values (SUV) on FDG-PET imaging (21;Fig. 3C and D).

Discussion

Few FHD studies of ASOs have sought to demonstratePD changes in cancer patients at safe doses such as reduc-tion of the targeted mRNA and protein levels in tumortissue (22–25). This FHD study of the second-generationASO LY2181308 was designed to include analysis of serialtumor samples to prove the concept that the ASO was ableto inhibit the function of survivin at a safe dose andschedule. We show that the ASO accumulated in tumortissue, significantly downregulated tumor survivin mRNA

and protein expression and enhanced the expression ofmarkers indicative of restored tumor cell apoptosis.

Consistent with previous studies, serial tumor biopsyprocedures were safe and accepted by patients (26). Ana-lysis of pre- and posttreatment biopsies demonstrated areduction of tumor mRNA and protein expression byapproximately 20% in a wide range of tumor types. Thisreduction is lower than that seen in xenograft models,where up to 50% reduction in survivin mRNA and proteinwere reported (10). In 5 cases, the presence of melanin,bilirubin, or hemosiderin in tumor tissue interfered withthe IHC staining and consequently changes in survivinlevels after treatment with LY2181308 could not be deter-mined. Tumor samples obtained from patients withNSCLC using fiberoptic bronchoscopy were perhaps evenmore effective in demonstrating survivin reduction. How-ever, a larger number of patients would need to be includedin future studies to confirm our observation. Althoughsurvivin levels were reduced, objective clinical responses

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Fig. 3. Apoptosis pathwayrestoration and PD responses inpatients receiving 750 mgLY2181308. IHC detection of CC3(A) and Ki67 (B) in tumor tissueobtained pre- and posttreatmentwith LY2181308. Percentagechange from baseline isrepresented with medians andinterquartile ranges. [18F]FDG-PET images (40–60 minutes)uptake scaled between 0 and 17g/mL (SUV): prior to first loadingdose (C); and following themaintenance dose on day 22 (D).Inserts highlight change in uptakein mesothelioma tumor (samesubject as Fig. 2F and G).

Talbot et al.





were not observed and thus direct cytotoxic effects ontumors were difficult to determine. This was an expectedfinding in the context of a FHD study and because pre-clinical data suggested that LY2181308 has a cytostatic,rather than a cytotoxic, antitumor effect. Supporting thismode of action were the observations that 1 patient withmetastatic melanoma had stable disease while receivingmaintenance LY2181308 weekly for 18 months, and 1patient who had a partial metabolic tumor response basedon FDG-PET imaging. Although antitumor efficacy was nota primary endpoint of the study, it was disappointing thatno objective responses were seen. Whether this would havebeen achieved had survivin mRNA and/or protein levelsbeen reduced by more than 20% cannot be addressed bythe current study. Combination studies of LY2181308 willassess whether inhibition of survivin enhances antitumoreffects of proapoptotic agents such as docetaxel.An important goal of this study was to demonstrate that

PD responses were consistent with the predicted PK profileof LY2181308 in plasma and tissue (Table 3) and itssimilarity with other second-generation ASOs (9). Thisprediction was confirmed at the 750 mg dose (Table 3).This is consistent with the observation that ASOs can bescaled successfully from animal to human. The PK profile ofLY2181308 and its terminal half-life of about 30 daysrequire a weekly maintenance dose to keep tissue concen-trations at levels that were associated with target inhibitionin animals. We also demonstrate that the ASO penetratestumor tissue as detectedby IHCandquantitative assessmentof ASO levels by ELISA (Table 3). The simulated concentra-tions in the tumor tissue were confirmed by concentrationmeasurements using ELISA and the [11C]LY2181308 study(Table 3). The range of LY2181308 concentration levelobserved in the tumor is slightly lower though overlappingrelative to the range of LY2181308 through plasma con-centration. This could be explained by the fact that othertissues, such as liver and kidney, in addition to the tumor,contribute to the equilibrium between LY2181308 tissuesand plasma concentrations The advantage of employingIHC is that it was possible to localize both the disposition ofthe ASO within tumor tissue and its intracellular distribu-tion. There appeared to be a difference in localization of theASO within tumor cells and the tumor microenvironment.Whether this observation was a result of the wide range oftumor types studied, the site of the biopsy or the timing afterthe last dose of LY2181308 is unclear (15 patients hadtheir biopsy taken on day 4; 8 on day 5; 1 on day 6; and2 on day 7). The variance in timing of posttreatmentbiopsies resulted in a degree of heterogeneity of resultsand may have introduced bias. Future studies shoulddefine consistent timing of biopsies for all patients andstudy sites. The use of [11C]LY2181308 PET further sup-ported the IHC-based observation that the ASO penetratedtumor tissue at a pharmacologically relevant concentration.Hence, LY2181308 clearly accumulated in tumor tissue overtime (17). This is consistent with studies in animals, wherethe distribution and stability of the second-generationASOswas evaluated (9, 27). Other FHD studies evaluating inhi-

bitors of the IAP family have not reported similar PDchanges in solid tumor tissue after dosing with ASOs. Forexample, the small molecular weight survivin inhibitorYM155 was only evaluated for safety and PKs (28), whereasin the clinical trial of the X-linked IAP (XIAP) ASO inhibitorAEG35156 PD changes in PBMCs (peripheral bloodmono-nuclear cells) were reported in addition to safety and PKs(25). Furthermore, the first-generationASOoblimersenwasevaluated for PDactivity inmelanomapatients only, but theproposed dose and dose schedule was not chosen for futurephase 2 studies (22). The second-generation ASOOGX-011against clusterin did evaluate PD changes in prostate cancertumor tissue and is the only trial that established a dose anddose regimen based on PD activity in cancer patients thatwas later used in phase 2 studies (23).

Finally, the dose range at which we observed consistentsurvivin reduction in tumor tissue had a favorable toxicityprofile for future clinical development. Grade 3 and 4 toxi-cities were present in 11 patients (27.5%) and acute renalfailurewasnotobservedduring the first cyclesof treatmentasreported for YM-155, a small molecule survivin inhibitor(28). However, we did observe increased creatinine values(grade2),which returned tobaseline levels after stopping theagent in 1 patient with metastatic melanoma who receivedLY2157299at the750mgdose for18months (29).Althoughseveral confounding factors were present, we cannot excludethatLY2157299treatmentwasassociatedwith this reversiblerenal injury. Although not considered medically adverse,lymphopenia was seen in 70% of the patients. Whether thiswas aPDeffect of LY2181308-induced apoptosis of survivin-expressinglymphocytes(30)orreflectedanoff-targeteffectoftheASOwill require additional investigation.A similar effecton lymphocyte counts was seen with the ASO AEG35156against XIAP (25). Typical off-target effects of ASO adminis-tration were observed for LY2181308 including anemia,thrombocytopenia, and transient prolongation in aPTT(31). In contrast to the phosphothioate ASOs or high dosesof second-generation ASOs, the off-target toxicity ofLY2181308 was milder and generally limited to grade 1and 2. This favorable toxicity profile was also recently con-firmed in Japanese patients, in particular the loading dose-associated elevation of complement Bb (32).

In conclusion, the integration of PD and PK analyses inthis FHD study has provided the proof of concept ofeffective and specific downregulation of the key molecu-lar target, survivin, in tumor tissue by the second-genera-tion ASO, LY2181308. These findings validate theapplication of second-generation ASOs for cancerpatients. The 750-mg dose and schedule of LY2181308is currently being evaluated in clinical studies in combi-nation with agents that induce apoptosis such as che-motherapy or radiation (33, 34).

Disclosure of Potential Conflicts of Interest

The authors M. Ranson, S. Callies, V. Andr!e, S. Kadam, M. Burgess, andC. Slapak are employees of Eli Lilly and Company, are fully compensated,and hold stock in the company. D. C. Talbot has received other commercialresearch support, honoraria from Speaker’s Bureau, and is on the Advisory






Board for Eli Lilly and Company. The other authors declare no potentialconflicts of interest.

Acknowledgments

We thank Drs. C. Prenant, G. Brown, T. Jones, and A. McMahon for[11C]LY2183108 radiolabeling, Dr. M. Slade (Oxford Radcliffe Hospitals)for endobronchial tumor sampling, B. Monia (Isis) for review of themanuscript, and J. Grimes, J. Birkett, C. Leppert, C. Stoner, Helen Desmierand Stacey Maxwell for data validation and study report writing.

Grant Support

The study received funding from Cancer Research UK and the ExperimentalCancer Medicine Centres of Oxford, Manchester and London. The study wassponsored by Eli Lilly and Company.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received 07/22/2010; revised 09/27/2010; accepted 10/07/2010;published OnlineFirst 11/01/2010.

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Talbot et al.





Correction

Correction: Tumor Survivin Is Downregulated bythe Antisense Oligonucleotide LY2181308: AProof-of-Concept, First-in-Human Dose Study

In this article (Clin Cancer Res 2010;16:6150–8), which was published in theDecember 15, 2010 issue of Clinical Cancer Research (1), the following statementshould have been included in the Grant Support section: "D.C. Talbot issupported by the NIHR Oxford Biomedical Research Centre."

Reference1. Talbot DC, Ranson M, Davies J, Lahn M, Callies S, Andr�e V, et al. Tumor survivin is downregulated

by the antisense oligonucleotide LY2181308: a proof-of-concept, first-in-human dose study. ClinCancer Res 2010;16:6150–8.

Published OnlineFirst April 12, 2011.�2011 American Association for Cancer Research.doi: 10.1158/1078-0432.CCR-11-0517

ClinicalCancer

Research

Clin Cancer Res; 17(8) April 15, 20112602

2010;16:6150-6158. Published OnlineFirst November 1, 2010.Clin Cancer Res Denis C. Talbot, Malcolm Ranson, Joanna Davies, et al. LY2181308: A Proof-of-Concept, First-in-Human Dose StudyTumor Survivin Is Downregulated by the Antisense Oligonucleotide

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