A Proof-Of-Concept, First-In-Human Dose Study

Published OnlineFirst November 1, 2010; DOI: 10.1158/1078-0432.CCR-10-1932

Clinical Cancer Cancer Therapy: Clinical Research

Tumor Survivin Is Downregulated by the Antisense Oligonucleotide LY2181308: A Proof-of-Concept, First-in-Human Dose Study

Denis C. Talbot1, Malcolm Ranson2, Joanna Davies1, Michael Lahn3, Sophie Callies4, Valerie Andre4, Sunil Kadam3, Michael Burgess4, Christopher Slapak3, Anna L Olsen1, Peter J. McHugh1, Johann S. de Bono5, Julian Matthews6, Azeem Saleem6, and Patricia Price7

Abstract Purpose: 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 essential gene BIRC5, was originally identified as an inhibitor of Authors' Affiliations: 1Department of Medical Oncology, University of apoptosis (IAP) that exerts its effects through binding to Oxford, Oxford Radcliffe Hospitals NHS Trust, Oxford, 2School of Cancer and Enabling Sciences, MAHSC, University of Manchester, Christie Hos- SMAC (second mitochondrial activator of caspases) pre- pital NHS Foundation Trust, Manchester, United Kingdom; 3Early Oncol- venting the sequestration of IAPs by SMAC and inhibition of 4 ogy Clinical Investigation, Eli Lilly & Co, Indianapolis, Indiana; and Eli Lilly caspase-dependent apoptosis (1). As a component of the & Co, Erl Wood Research Centre, Windlesham, 5Institute of Drug Devel- opment, Royal Marsden, Sutton, 6Wolfson Molecular Imaging Centre, kinetochore-associated complex, survivin also plays an MAHSC, University of Manchester, and 7Academic Radiation Oncology, important role in the regulation of late mitosis and cytokin- The Christie Hospital, National Health Services (NHS) Foundation Trust, University of Manchester, Manchester, United Kingdom esis (2). Survivin is expressed in a wide range of human cancers and when overexpressed, is associated with a poorer 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. prognosis (3). With the exception of placenta, thymus, First human dose study evaluating safety and pharmacokinetics of activated T cells, gastrointestinal crypt cells, and regenerat- LY2181308, an antisense oligonucleotide designed to inhibit survivin. ASCO Abstract 3518, Oral Presentation, Developmental Therapeutics: ing liver, survivin is not expressed in normal adult tissue (4). Molecular Therapeutics, June 1, 2008. Thus, survivin represents an attractive molecular target for D.C. Talbot, J. Davies, A.L. Olsen, V. Andre, M. Lahn, E. Powell, S. Kadam, therapeutic intervention. Targeted approaches against sur- J. De Bono, P.J. McHugh, M. Ranson. Pharmacodynamic evaluation of vivin include small molecule inhibitors against the survivin LY2181308 in Patients with metastatic malignancies. Abstract 3507, Oral protein, gene silencing, and survivin mRNA blockade (5, 6). Presentation, Clinical Science Symposium, May 30, ASCO 2009. The 20-O-methoxyethyl–modified ASO (second generation) Corresponding Author: Denis C. Talbot, Department of Medical Oncology, University of Oxford, Cancer and Haematology Center, Churchill Hospital, LY2181308 is an 18-mer ASO that binds to the translation Old Road, Headington, Oxford OX3 7LJ, United Kingdom. Phone: 44 (0)1865 initiation codon of the survivin transcript. Following ASO 235312; Fax: 44 (0)1865 235985; E-mail: [email protected]. hybridization to survivin mRNA, RNase H–dependent clea- doi: 10.1158/1078-0432.CCR-10-1932 vage of the duplex ensues with subsequent degradation of 2010 American Association for Cancer Research. the survivin mRNA (7). Thus, survivin protein expression is

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Translational Relevance reduction in survivin expression in tumor tissue at the anticipated BED as defined by the preclinical PK/PD model Evasion of apoptosis is a hallmark of malignancy. of Callies et al. (10). At the recommended phase 2 dose, 2 Survivin is a small, naturally occurring inhibitor of companion studies were conducted at the Universities of apoptosis (IAP) that is often highly expressed by cancer Oxford (study 1, endobronchial tumor sampling) and 11 cells making it an attractive target for therapeutic Manchester (study 2, [ C]LY2181308 study). The entire intervention. The antisense oligonucleotide (ASO) study was approved by the Medicines and Healthcare LY2181308 downregulates survivin by targeting the products Regulatory Agency (MHRA) and a Multicentre survivin transcript and has antitumor efficacy through Research Ethics Committee (MREC). induction of tumor cell apoptosis in preclinical models. This First-In-Human (FHD) study demonstrates the Enrollment criteria proof of concept that ASO therapy directed against Inclusion criteria were as follows: at least 18 years of age, survivin mRNA reduces survivin mRNA and protein confirmed malignancy, exhausted approved standard thera- levels and restores apoptosis in tumors of cancer pies, tumor accessible for biopsy, written informed consent, patients. The research represents a critical step in the Eastern Cooperative Oncology Group (ECOG) perfor- translation from discovery of survivin, an important mance status of 0 or 1, discontinued previous anticancer 9 tumor-related IAP, to successful clinical application of therapies, absolute neutrophil count 1.5 10 /L, plate- 9 survivin-targeted therapy. Finally, this FHD study helps lets 100 10 /L, hemoglobin 9 g/dL, normal serum < to better understand how ASOs can be used as novel bilirubin 2.5 upper limit of normal, alanine transami- agents in the treatment of cancer. nase (ALT) and aspartate transaminase (AST), calculated creatinine clearance 50 mL/min, normal activated prothrombin time (aPTT), and prothrombin time (PT), and contraceptive precautions taken. Exclusion criteria were as specifically inhibited without affecting expression of other follows: bleeding diathesis, major surgery within last 4 genes including other IAP proteins (8). Compared with weeks, pregnant or lactating, symptomatic central nervous phosphorothioate or first-generation ASOs, second-genera- system (CNS) neoplasm, taking concomitant anticoagulant tion ASOs are more stable, have an improved pharmaco- therapy, received prior ASO, treatment with an unapproved kinetic (PK) profile, increased potency, and reduced toxicity drug, positive test results for viral infection. (9). Using aggregate data from preclinical pharmacology and toxicology, an integrated PK/PD (pharmacodynamic) Drug formulation and schedule of administration model was developed to predict a biologically effective dose LY2181308 was diluted in 500 mL of normal saline for range for clinical evaluation (10). On the basis of this, a first- intravenous injection and infused as a 3-hour infusion on 3 in-human dose (FHD) study was designed to evaluate the consecutive days as a loading dose, followed by weekly biodistribution profile of LY2181308 by measuring the maintenance doses. The dose and dose schedule were based following tumor-specific PD changes: tumor tissue penetra- on preclinical safety, PK and pharmacology studies of tion of LY2181308, downregulation of tumor survivin at the LY2181308. These were integrated within a PK/PD model mRNA, and protein levels and restoration of tumor apop- that predicted the BED (10). tosis at a dose and schedule that was safe in humans. To this end, pre- and posttreatment tumor sampling was per- Treatment assessment formed for rigorous assessment of target modulation (i.e., DLT was defined (CTCAE Version 3.0; ref. 11) as 1 of 3 survivin protein and gene expression), the effect of restoring patients with the following: grade 3/4 hematologic toxicities normal apoptosis/cell cycle–related protein activity and more than 5 days, aPTT prolongation more than 48 hours biodistribution of LY2181308 including subcellular locali- after infusion, any grade 3/4 nonhematologic toxicity or any zation. other DLT including those associated with sustained complement activation. Laboratory examinations: C-reactive Patients and Methods protein (CRP), complement split products (National Jewish Medical and Research Center), hematology, serum chem- Study design istry. Pretreatment tumor biopsy: after enrollment and This FHD monotherapy study was divided into 3 parts: before first loading. Posttreatment tumor biopsy: window part A (safety, PK), 1-patient cohorts with initial 100 mg of 48 to 96 hours after the third loading dose. Descriptive and escalated, by dose doubling, to 400 mg; part B (safety, statistics were used for all patients receiving at least 1 dose of PK, PD), 3-patient cohorts with 50% dose escalation until LY2181308 to evaluate safety, PK, and efficacy. dose-limiting toxicity (DLT); part C (dose confirmation cohort, PK, PD). The transition from part A to part B was Pharmacokinetic assessment planned on the basis of either development of toxicity or PK parameters were analyzed (WinNonlin Enterprise, evidence from PK that the predicted biological effective Version 5.2) for: time of maximum concentration (Tmax), dose (BED) had been reached. The recommended dose for maximum plasma concentration (Cmax), area under phase 2 studies was determined from both DLT and the the plasma concentration versus time curve (AUC), and

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clearance (CL) following the third and fifth administration Results of LY2181308 on days 3 and 15. Plasma PK data were pooled for nonlinear mixed affect modeling analysis (NON- Between October 2004 and December 2008, 40 patients MEM, Version 6.1) to determine the compartmental PK were enrolled in this monotherapy FHD study, of whom 17 parameters: mean value, variance, and subject variability. were enrolled in the initial dose-escalation stage with doses The interindividual variability was coded as an exponential ranging from 100 mg to 1,000 mg. Twenty-six patients were model and the residual variability as a proportional model. treated at the 750 mg dose level, including 6 patients in 2 site-specific studies: study 1 assessed apoptosis and cell- Development of a specific antibody to detect the ASO cycle progression changes by flow cytometry of endobron- A polyclonal rabbit antibody against the ASO was gen- chial tumor cells in 3 patients with NSCLC (non–small cell erated using a keyhole limpet hemocyanin (KLH)–modified lung carcinoma); study 2 examined the biodistribution of ASO (Lampire Biological Laboratories). It was validated for [11C]LY2181308 measured by PET in 3 patients. The demo- use in formalin-fixed, paraffin-embedded specimen utiliz- graphics of the study population was typical of phase 1 ing Ventana’s DiscoveryXTstaining platforms (Ventana). oncology trials (19) with the majority of patients having received prior chemotherapy (Table 1). Fourteen patients Pharmacodynamic assessment with hepatic metastasis were included, of whom 11 had Formalin-fixed tissue was used for immunohistochem- biopsies taken from liver lesions. LY2181308 was well ical assessment at Ventana (Tucson) to determine: ASO, tolerated with the majority of the patients showing grade survivin (NB500-201; Novus Biologicals), cleaved caspase 1 or 2 toxicities (29/40, 72.5%). Of the 40 patients, 11 3 (CC3; CST9661; Cell Signaling Technologies), and Ki67 patients (27.5%) had grade 3 or 4 toxicities (Table 2). Two (mouse monoclonal antibody; Ventana). All antibodies of the 11 patients received the 1,000 mg dose. One patient were detected by appropriate secondary antibodies (Vector had a grade 3 hypophosphatemia, whereas the other Laboratories). Staining intensity was determined using the patient experienced a grade 4 lymphopenia and a pro- HSCORE and Ventana’s automated imaging software (12). longed Grade 3 headache, which did not respond to pain Gene expression analysis (Panomics; ref. 13) was used to medication. This event was defined as the DLT for this quantify Survivin mRNA expression (branched DNA, study. Concurrently, a sharp rise in CRP was observed in bDNA). Percentage change in protein and mRNA expres- this patient raising the concern of a possible complement- sion was summarized using medians and approximately 95% confidence limits (14). Table 1. Baseline patient and disease charac- teristics (n ¼ 40) Endobronchial biopsy Fiberoptic endobronchial tumor sampling is a safe, Sex n (%) short, and standard procedure (15). Endobronchial tumors Male 19 (47.5) were visualized and sampled by brushing with a 1-mm Female 21 (52.5) brush. The samples were processed immediately for flow Age group cytometry assessment. <65 32 (80.0) >65 8 (20.0) Flow cytometry ECOG performance status Briefly, disaggregated tumor cells were fixed in formal- 0 15 (37.5) dehyde and subsequently stained with phycoerytherin- 1 25 (62.5) (PE)conjugated monoclonal antibodies directed against Pathologic diagnosis (n) survivin (Clone 91630; R&D Systems). Flow cytometric Gastrointestinal tumors 12 (30.0) data (obtained on a CyAN machine, Dako) were analyzed (including 7 colon, using Summit software (Dako). 1 rectal, 2 gastric, 1 esophageal, 11 [ C]LY2181308 PET imaging 1 pancreas cancer) After establishing safety in nonhuman primates (16), Breast cancer 8 (20.0) patients received less than 1 mg and less than 600 MBq of Melanoma 7 (17.5) 11 [ C]LY2181308 prior to LY2181308, and during the Lung cancer 7 (17.5) maintenance infusion on day 15 (17). PET data were Other (3 sarcoma, 6 (15.0) collected for 90 minutes following bolus radiotracer 1 ovary, 1 head injection. and neck, 1 unknown adenocarcinoma) FDG-PET imaging Prior therapy FDG-PET imaging was performed prior to treatment with Radiotherapy 23 (57.5) LY2181308 and on day 22. The imaging was performed as Surgery 30 (75.0) previously recommended (18) and consistent with institu- Chemotherapy 39 (97.5) tional radiation guidelines.

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

CTCAE description Maximum CTC grade

1 234

Laboratory Partial thromboplastin time 28 2 Platelet counts 7 2 ALT/SGPT (serum glutamic pyruvic transaminase) 5 2 Metabolic/laboratory—other (including CRP) 5 1 Hemoglobin 3 2 Lymphopenia 2 1 1 1 AST/SGOT (serum glutamic oxaloacetic transaminase) 3 1 Phosphate (hypophosphatemia) 1 3 Potassium (hypokalemia) 4 Glucose (hyperglycemia) 2 1 Leukocytes (total WBC) 3 Blood/bone marrow—other (eosinophils, etc) 2 1 Alkaline phosphatase 1 1 Bilirubin (hyperbilirubinemia) 2 Sodium (hyponatremia) 1 1 Nonlaboratory Fever (in the absence of neutropenia) 9 4 1 Nausea 7 3 1 Fatigue (asthenia, lethargy, malaise) 6 3 1 Vomiting 6 1 Diarrhea 2 1 Pain neurology—head/headache 2 1 Rigors/chills 2 1 Flu-like syndrome 1 1 Hypotension 1 1 Pain musculoskeletal-–joint 2 Sweating (diaphoresis) 2

Abbreviation: CTC, circulating tumor cells. induced cerebrospinal leak syndrome. Three patients were The PK properties of LY2181308 and reduction of treated at the 900 mg dose level and no DLTs were survivin expression in tumor tissue were assessed by observed, but CRP, AST/ALT, and moderate flu-like symp- collecting plasma samples and tumor biopsies before toms occured during the loading doses, suggesting con- and after completing the loading dose. In parts B and tinued complement-activation. The most common C of the protocol, 31 patients were enrolled with 26 symptoms were flu-like syndrome (fever, rigor, musculos- (84%) agreeing to have both pre- and posttreatment keletal pain, nausea), fatigue, and vomiting (Table 2). The biopsies. Ten patients had either insufficient tumor mate- most frequent laboratory toxicity was prolongation of aPTT rial in the posttreatment biopsy or endogenous pigments (generally grade 1), which was observed at the time of the (bilirubin, hemosiderin, and melanin) that interfered infusion in 75% of the patients. Other laboratory abnorm- with IHC staining. Hence, in 16 (52%) patients, we alities, not considered as severe adverse events by investi- obtained sufficient pre- and postdosing tumor tissue to gators, included lymphopenia (70%), thrombocytopenia assess the PD effects of LY2181308 by protocol-defined (38%), hypokalemia (38%), and anemia (35%). Transient laboratory procedures (Fig. 1A–F). increases in complement Bb were noted at the end of the Survivin protein expression as measured by IHC was third loading dose, but was not associated with adverse reduced by 21%, concomitant with a statistically significant clinical events. Given the comparable PK profile of the 20% reduction in mRNA expression (P < 0.05) as shown by higher doses of 900 and 1,000 mg to the 750-mg dose, the gene expression analysis (Fig. 1G). Because of the high recommended dose of LY2181308 for further clinical stu- intensity of nuclear survivin IHC staining prior to the dies using the same dosing schedule was defined as 750 mg, treatment, reduction of nuclear protein expression was a dose likely to be safe in combination with chemother- more clearly detected than changes seen in the cytoplasm apeutics. (Fig. 1E–G). Similarly, in the site-specific study in NSCLC, a

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A C E low high H

Pre Pre

10o 101 102 103 104

B D F C low high

Post Post

10o 101 102 103 104 Survivin(PE) G I

40 Pretreatment Posttreatment 30

-50 0 50 100 0 % high -survivin staining

Percent change from baseline Patient 1 Patient 2 Patient 3 -100

mRNA Nucleus (HC) Cytoplasm (IHC)

Fig. 1. Reduction of survivin protein and mRNA expression after loading dose of LY2181308. IHC of tumor biopsy from 1 representative patient with breast cancer (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. Flow cytometric analysis of survivin expression by endobronchial NSCLC obtained from 3 patients by fiberoptic-guided bronchial brushing prior to and after LY2181308 (H) and change in high survivin-expressing cells (I). PE, phycoerythrin.

reduction of survivin protein expression was demonstrated following administration of 1 mg of [11C]LY2181308, by flow cytometry in endobronchial tumor cells compared tumor penetration of LY2181308 ranged from 21 to 84 ng with pretreatment levels in all 3 patients studied (Fig. 1I). h/mL tumor with a maximum concentration that ranged The PK of LY2181308 confirmed a multiphasic dispo- from 10 to 60 ng/mL. These levels are comparable with sition in plasma with rapid tissue distribution and half- the concentration measured by ELISA in tumor biopsy lives of 0.5, 2.5, and 12 hours, and an elimination half- and with that required for target inhibition (Table 3). The life of 31 days (Fig. 2A). The terminal half-life was the PK profile and tissue distribution of LY2181308 were result of a clearance of 20 to 30 L/h and volume of consistent with the PD effects of the agent described distribution of more than 1,000 L consistent with the above. However, there was no evident relationship tissue distribution of LY2181308 of approximately 90%. between the localization of the ASO within the tumor As described for other ASOs (9), the primary sites of tissue compartments, clinical response, or change in normal tissue uptake of LY2181308 were renal and hepa- survivin expression, perhaps due to the limited numbers tic, with less uptake seen in other normal tissue (17). The of patients included within each cohort. ASO was detected by IHC in tumor tissue in 10 of 11 Of 22 patients assessed for efficacy using the RECIST patients, either within tumor cells or tumor-associated (response evaluation criteria in solid tumors) criteria (20), macrophages (Fig. 2B and C) and in 5 of 11 patients in a total of 4 patients achieved stable disease, including 1 stroma(Fig.2DandE).CompatiblewiththeIHCstain- patient with metastatic melanoma who remained free of ing, the [11C]LY2181308 PET imaging confirmed that disease progression for 18 months. Reduction of survivin

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10 6 A B D

10 5 Pre

10 4

10 3 C E LY2181308 (ng/mL) 10 2 Post

10 1 10/11 5/11 0 24 48 72 96 120 144 168 192 216 240 312 336 360 384 408 432 456 480 504 528 h

F G

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 representative patients (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 the 11 patients showing a similar pattern (D, E). Tissue PKs assessed by [11C]LY2181308 uptake (AUC during 90 minutes scan) scaled between the 0 and 140 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 change in uptake in specific area of mesothelioma over time.

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

Predicted Observed 1,000 simulations n ¼ 24

Plasma PK

AUC0–24, 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 PKs Concentration, 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).

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A 50 B 0

Fig. 3. Apoptosis pathway restoration and PD responses in patients receiving 750 mg 100 200 300 400 LY2181308. IHC detection of CC3 Percent change from baseline Percent change from baseline (A) and Ki67 (B) in tumor tissue obtained pre- and posttreatment with LY2181308. Percentage 0 change from baseline is

-100 -50 represented with medians and 18 CC3 nucleus (IHC) KI67 nucleus (IHC) interquartile ranges. [ F]FDG- PET images (40–60 minutes) uptake scaled between 0 and 17 g/mL (SUV): prior to first loading dose (C); and following the C D maintenance dose on day 22 (D). Inserts highlight change in uptake in mesothelioma tumor (same subject as Fig. 2F and G).

expression was associated with an increase in the apoptosis and protein expression and enhanced the expression of marker CC3 and a reduction in tumor expression of the markers indicative of restored tumor cell apoptosis. proliferation marker Ki67 (Fig. 3A and B). In the single Consistent with previous studies, serial tumor biopsy patient assessed, a partial metabolic response in several procedures were safe and accepted by patients (26). Ana- mesothelioma lesions was observed with a 40% reduction lysis of pre- and posttreatment biopsies demonstrated a in standard uptake values (SUV) on FDG-PET imaging (21; reduction of tumor mRNA and protein expression by Fig. 3C and D). approximately 20% in a wide range of tumor types. This reduction is lower than that seen in xenograft models, Discussion where up to 50% reduction in survivin mRNA and protein were reported (10). In 5 cases, the presence of melanin, FewFHDstudiesofASOshavesoughttodemonstrate bilirubin, or hemosiderin in tumor tissue interfered with PD changes in cancer patients at safe doses such as reduc- the IHC staining and consequently changes in survivin tion of the targeted mRNA and protein levels in tumor levels after treatment with LY2181308 could not be deter- tissue (22–25). This FHD study of the second-generation mined. Tumor samples obtained from patients with ASO LY2181308 was designed to include analysis of serial NSCLC using fiberoptic bronchoscopy were perhaps even tumor samples to prove the concept that the ASO was able more effective in demonstrating survivin reduction. How- to inhibit the function of survivin at a safe dose and ever, a larger number of patients would need to be included schedule. We show that the ASO accumulated in tumor in future studies to confirm our observation. Although tissue, significantly downregulated tumor survivin mRNA survivin levels were reduced, objective clinical responses

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were not observed and thus direct cytotoxic effects on bitors of the IAP family have not reported similar PD tumors were difficult to determine. This was an expected changes in solid tumor tissue after dosing with ASOs. For finding in the context of a FHD study and because pre- example, the small molecular weight survivin inhibitor clinical data suggested that LY2181308 has a cytostatic, YM155 was only evaluated for safety and PKs (28), whereas rather than a cytotoxic, antitumor effect. Supporting this in the clinical trial of the X-linked IAP (XIAP) ASO inhibitor mode of action were the observations that 1 patient with AEG35156 PD changes in PBMCs (peripheral blood mono- metastatic melanoma had stable disease while receiving nuclear cells) were reported in addition to safety and PKs maintenance LY2181308 weekly for 18 months, and 1 (25). Furthermore, the first-generation ASO oblimersen was patient who had a partial metabolic tumor response based evaluated for PD activity in melanoma patients only, but the on FDG-PET imaging. Although antitumor efficacy was not proposed dose and dose schedule was not chosen for future a primary endpoint of the study, it was disappointing that phase 2 studies (22). The second-generation ASO OGX-011 no objective responses were seen. Whether this would have against clusterin did evaluate PD changes in prostate cancer been achieved had survivin mRNA and/or protein levels tumor tissue and is the only trial that established a dose and been reduced by more than 20% cannot be addressed by dose regimen based on PD activity in cancer patients that the current study. Combination studies of LY2181308 will was later used in phase 2 studies (23). assess whether inhibition of survivin enhances antitumor Finally, the dose range at which we observed consistent effects of proapoptotic agents such as docetaxel. survivin reduction in tumor tissue had a favorable toxicity An important goal of this study was to demonstrate that profile for future clinical development. Grade 3 and 4 toxi- PD responses were consistent with the predicted PK profile cities were present in 11 patients (27.5%) and acute renal of LY2181308 in plasma and tissue (Table 3) and its failure wasnotobserved during the first cycles of treatmentas similarity with other second-generation ASOs (9). This reported for YM-155, a small molecule survivin inhibitor prediction was confirmed at the 750 mg dose (Table 3). (28). However, we did observe increased creatinine values This is consistent with the observation that ASOs can be (grade 2),which returned to baseline levels afterstopping the scaled successfully from animal to human. The PK profile of agent in 1 patient with metastatic melanoma who received LY2181308 and its terminal half-life of about 30 days LY2157299atthe750mgdose for18months (29).Although require a weekly maintenance dose to keep tissue concen- several confounding factors were present, we cannot exclude trations at levels that were associated with target inhibition that LY2157299treatmentwasassociated with this reversible in animals. We also demonstrate that the ASO penetrates renal injury. Although not considered medically adverse, tumor tissue as detected by IHC and quantitative assessment lymphopenia was seen in 70% of the patients. Whether this of ASO levels by ELISA (Table 3). The simulated concentra- was a PD effect of LY2181308-induced apoptosis of survivin- tions in the tumor tissue were confirmed by concentration expressinglymphocytes(30)orreflectedanoff-targeteffectof measurements using ELISA and the [11C]LY2181308 study the ASO will require additional investigation. A similar effect (Table 3). The range of LY2181308 concentration level on lymphocyte counts was seen with the ASO AEG35156 observed in the tumor is slightly lower though overlapping against XIAP (25). Typical off-target effects of ASO adminis- relative to the range of LY2181308 through plasma con- tration were observed for LY2181308 including anemia, centration. This could be explained by the fact that other thrombocytopenia, and transient prolongation in aPTT tissues, such as liver and kidney, in addition to the tumor, (31). In contrast to the phosphothioate ASOs or high doses contribute to the equilibrium between LY2181308 tissues of second-generation ASOs, the off-target toxicity of and plasma concentrations The advantage of employing LY2181308 was milder and generally limited to grade 1 IHC is that it was possible to localize both the disposition of and 2. This favorable toxicity profile was also recently con- the ASO within tumor tissue and its intracellular distribu- firmed in Japanese patients, in particular the loading dose- tion. There appeared to be a difference in localization of the associated elevation of complement Bb (32). ASO within tumor cells and the tumor microenvironment. In conclusion, the integration of PD and PK analyses in Whether this observation was a result of the wide range of this FHD study has provided the proof of concept of tumor types studied, the site of the biopsy or the timing after effective and specific downregulation of the key molecu- the last dose of LY2181308 is unclear (15 patients had lar target, survivin, in tumor tissue by the second-genera- their biopsy taken on day 4; 8 on day 5; 1 on day 6; and tion ASO, LY2181308. These findings validate the 2 on day 7). The variance in timing of posttreatment application of second-generation ASOs for cancer biopsies resulted in a degree of heterogeneity of results patients. The 750-mg dose and schedule of LY2181308 and may have introduced bias. Future studies should is currently being evaluated in clinical studies in combi- define consistent timing of biopsies for all patients and nation with agents that induce apoptosis such as che- study sites. The use of [11C]LY2181308 PET further sup- motherapy or radiation (33, 34). ported the IHC-based observation that the ASO penetrated tumor tissue at a pharmacologically relevant concentration. Disclosure of Potential Conflicts of Interest Hence, LY2181308 clearly accumulated in tumor tissue over time (17). This is consistent with studies in animals, where The authors M. Ranson, S. Callies, V. Andr!e, S. Kadam, M. Burgess, and C. Slapak are employees of Eli Lilly and Company, are fully compensated, the distribution and stability of the second-generation ASOs and hold stock in the company. D. C. Talbot has received other commercial was evaluated (9, 27). Other FHD studies evaluating inhi- research support, honoraria from Speaker’s Bureau, and is on the Advisory

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Talbot et al.

Board for Eli Lilly and Company. The other authors declare no potential Grant Support conflicts of interest. The study received funding from Cancer Research UK and the Experimental Cancer Medicine Centres of Oxford, Manchester and London. The study was Acknowledgments sponsored by Eli Lilly and Company. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked We thank Drs. C. Prenant, G. Brown, T. Jones, and A. McMahon for advertisement [11C]LY2183108 radiolabeling, Dr. M. Slade (Oxford Radcliffe Hospitals) in accordance with 18 U.S.C. Section 1734 solely to indicate for endobronchial tumor sampling, B. Monia (Isis) for review of the this fact. manuscript, and J. Grimes, J. Birkett, C. Leppert, C. Stoner, Helen Desmier Received 07/22/2010; revised 09/27/2010; accepted 10/07/2010; and Stacey Maxwell for data validation and study report writing. published OnlineFirst 11/01/2010.

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6158 Clin Cancer Res; 16(24) December 15, 2010 Clinical Cancer Research

Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2010 American Association for Cancer Research. Clinical Cancer Correction Research Correction: Tumor Survivin Is Downregulated by the Antisense Oligonucleotide LY2181308: A Proof-of-Concept, First-in-Human Dose Study

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

Reference 1. Talbot DC, Ranson M, Davies J, Lahn M, Callies S, Andre V, et al. Tumor survivin is downregulated by the antisense oligonucleotide LY2181308: a proof-of-concept, first-in-human dose study. Clin Cancer Res 2010;16:6150–8.

Published OnlineFirst April 12, 2011. Ó2011 American Association for Cancer Research. doi: 10.1158/1078-0432.CCR-11-0517

2602 Clin Cancer Res; 17(8) April 15, 2011 Published OnlineFirst November 1, 2010; DOI: 10.1158/1078-0432.CCR-10-1932

Tumor Survivin Is Downregulated by the Antisense Oligonucleotide LY2181308: A Proof-of-Concept, First-in-Human Dose Study

Denis C. Talbot, Malcolm Ranson, Joanna Davies, et al.

Clin Cancer Res 2010;16:6150-6158. Published OnlineFirst November 1, 2010.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-10-1932

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