Apo2L/TRAIL-inducedapoptosis3, 15, while Apo2L/TRAIL. 0 did not (Fig. 2c, top). ViabilityassaysusingAlamarBlue, MTT, CrystalVioletandToludineBlue (datanot shown) confirmed minimalinductionof hepatocyte apoptosis or necrosis by Apo2L/TRAIL. 0. Consistentdata wereobtained at Genentech, Immunex and UniversityofPittsburgh, withhepatocytes fromeightdonors, isolatedeitherbytwo commercialsourcesorbyS. SS Thus, despiteisolationandcultureinlowserum, hepatocytes show little sensitivity to Apo2L/TRAIL. 0. Previously, wehaveusedthecynomolgus monkeyasapreclinicalsafety modelfor Apo2L/TRAIL (ref. 3). Apo2L/TRAILandits receptorsDR4, DR5, DcR2andOPG (ref. 16) showed84–99% extracellularproteinsequenceidentity between cynomolgus and humancounterparts (notshown). Cynomolgus DcR1 appearedto be a pseudo-gene; however, absenceofthis antagonisticreceptor, ifrelevant, might beexpectedtoincreasesensitivityto Apo2L/TRAIL. Human Apo2L/TRAIL. 0 boundwithcomparableaffinitytoFc-fusionproteinsofeachhumanorcynomolgusreceptor (notshown), demonstrating goodcross-reactivity. Asevidenced by morphology, DNAfragmentation, and PARP cleavage (Fig. 2a–c, bottom), Apo2L/TRAIL. His induced substantial apoptosisinisolatedcynomolgushepatocytes, whereasApo2L/TRAIL. 0had marginal effects, similar to results with humanhepatocytes. Notably, AlamarBlue or MTTassaywasnotadequatefordetectingdeathincynomolgushepatocytesdespiteclearevidenceofapoptosis, perhaps explaining whyrhesus hepatocytes appeared previously8 to be resistant to Apo2L/TRAIL. Hisby MTTassay. Neither TNF (0.1 µg/ml), norinterleukin-1β (0.1 µg/ml), norcycloheximide (0.5 µg/ml) sensitized cynomolgus hepatocytes to Apo2L/TRAIL. 0-induced DNAfragmentation (datanotshown), furtherdemonstrating minimaltoxicity of Apo2L/TRAIL. 0 towardthesecells. Bindingofradio-iodinatedApo2L/TRAIL. Histocynomolgushepatocytes wasnotdisplacedbyunlabeled ligand (Fig. 2d), suggestingirreversibleinteraction; bindingof Apo2L/TRAIL. 0 was displacedinadose-dependentfashion, indicatingreversibleassociation. Together, thesedatademonstratethatthecynomolgus monkeyisavalidspeciesforpreclinical safetyinvestigationofclinical-graderecombinanthumanApo2L/TRAIL. Intravenous (iv) administration of Apo2L/TRAIL. 0incynomolgus monkeys (0, 0.1, 1, or10 mg/kg/d× 7d, n= 3/dose) waswelltolerated3. Weassessedpotential hepatotoxicityinafurtherstudy, bydosing vehicle or Apo2L/TRAIL. 0 at 100 mg/kg/dover5d (n= 12/group). There werenonotabledifferencesbetweenthe groupsinclinicalsigns, foodconsumption, bodyweight, serumchemistry, coagulation parameters, hematology, and urinalysis; importantly, therewerenosignificant changes in liver enzyme activities, bilirubin or albumin in serum. Histology of all major organs including liver was normal. SerumApo2L/TRAIL. 0peakedat–1000µg/mlandremainedforover7h above0. 1µg/ml, adoseofApo2L/TRAIL. Histhatinducedmarkedhepatocyteapoptosis within4hinvitro. Wealsostudied Apo2L/TRAIL. 0inchimpanzees. Chimpanzee and human Apo2L/TRAIL, DR4, DR5, DcR1, DcR2 and OPG showed 97–99% extracellularsequenceidentity (datanotshown). Afteri. v. injectionat5 mg/kg (n= 3), Apo2L/TRAIL. 0peakedin chimpanzeeserumat∼ 130µg/mlandremainedabove0. 1 µg/mlfor> 6h; theanimalsshowed nosignificantchangesin clinicalpathology, includingliverenzyme activities. Thus, tworelevantnon-human primate modelsindicatethatsystemicadministrationofApo2L/TRAIL. 0tocancer patientsisunlikelytocause majortoxicity totheliverorotherorgans. Furtherpreclinical safety studies of clinical-grade Apo2L/TRAIL …