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Cancer Therapy: Preclinical

Liver Microvascular Injury and Thrombocytopeniaof Antibody–Calicheamicin Conjugates inCynomolgus Monkeys—Mechanism andMonitoringMagali Guffroy1, Hadi Falahatpisheh1, Kathleen Biddle2, John Kreeger2, Leslie Obert2,Karen Walters2, Richard Goldstein2, Germaine Boucher2, Timothy Coskran2,William Reagan2, Danielle Sullivan2, Chunli Huang2, Sharon Sokolowski2,Richard Giovanelli2, Hans-Peter Gerber3, Martin Finkelstein1, and Nasir Khan2

Abstract

Purpose: Adverse reactions reported in patients treated withantibody–calicheamicin conjugates such as gemtuzumab ozoga-micin (Mylotarg) and inotuzumab ozogamicin include throm-bocytopenia and sinusoidal obstruction syndrome (SOS). Theobjective of this experimental work was to investigate the mech-anism for thrombocytopenia, characterize the liver injury, andidentify potential safety biomarkers.

ExperimentalDesign:Cynomolgusmonkeyswere dosed intra-venously at 6 mg/m2/dose once every 3 weeks with a nonbindingantibody–calicheamicin conjugate (PF-0259) containing thesame linker-payload as gemtuzumab ozogamicin and inotuzu-mab ozogamicin. Monkeys were necropsied 48 hours after thefirst administration (day 3) or 3 weeks after the third adminis-tration (day 63).

Results: PF-0259 induced acute thrombocytopenia (up to 86%platelet reduction) with nadirs on days 3 to 4. There was noindication of effects on megakaryocytes in bone marrow or

activationof platelets inperipheral blood.Microscopic evaluationof liver from animals necropsied on day 3 demonstrated mid-zonal degeneration and loss of sinusoidal endothelial cells (SECs)associated with marked platelet accumulation in sinusoids. Liverhistopathology on day 63 showed variable endothelial recoveryand progression to a combination of sinusoidal capillarizationand sinusoidal dilation/hepatocellular atrophy, consistent withearly SOS. Among biomarkers evaluated, there were early andsustained increases in serum hyaluronic acid (HA) that correlatedwell with serum aspartate aminotransferase and liver microscopicchanges, suggesting that HAmay be a sensitive diagnostic markerof the liver microvascular injury.

Conclusions: These data support the conclusion thattarget-independent damage to liver SECs may be responsiblefor acute thrombocytopenia (through platelet sequestra-tion in liver sinusoids) and development of SOS. Clin CancerRes; 23(7); 1760–70. �2016 AACR.

IntroductionGemtuzumab ozogamicin (Mylotarg) and inotuzumab ozo-

gamicin are antibody–drug conjugates (ADCs) composed ofdifferent humanized mAbs and of the same linker and payload(1). The specific payload is a calicheamicin derivative that binds totheDNAminor groove and induces double-strandedDNAbreaks.It is conjugated to the antibodies via an acid-labile linker thatensures calicheamicin release at the acidic pH of lysosomes afterADC internalization into target-expressing cells (1). In terms of

target specificity, gemtuzumab ozogamicin binds to CD33, whichis expressed on both normal and malignant myeloid cells and ispresent on leukemic blasts in over 80% of patients with acutemyeloid leukemia (AML; ref. 2). Gemtuzumab ozogamicin wasgranted accelerated approval by the FDA in 2000 for the treatmentof patients over 60 years of age with relapsed CD33-positive AML(3), but was voluntarily withdrawn in 2010 when a postapprovalcommitment study failed to confirm clinical benefit and hepaticsinusoidal obstruction syndrome (SOS) became recognized as anadverse event (4–6). However, several recent clinical trials haveclarified the optimal conditions for gemtuzumab ozogamicin useand demonstrated benefits to specific patient populations (7, 8).Currently, gemtuzumab ozogamicin is still approved and avail-able in Japan. Inotuzumab ozogamicin specifically targets theCD22 antigen, which is expressed on the surface of immature andmature B cells and is present in 60% to 90% of B-lymphoidmalignancies (9). Inotuzumab ozogamicin is currently developedfor the treatment of acute lymphoblastic leukemia.

Adverse reactions reportedwithboth these drugs include throm-bocytopenia and hepatic injury. Thrombocytopenia with gemtu-zumabozogamicin, although inpart expected fromthe targetingofCD33-positive hematopoietic progenitor cells, showed prolonged

1Drug Safety Research and Development, Pfizer Inc., Pearl River, New York.2Drug Safety Research and Development, Pfizer Inc., Groton, Connecticut.3Oncology-Rinat Research & Development, Pfizer Inc., Pearl River, New York.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Magali Guffroy, Drug Safety Research and Develop-ment, Pfizer Inc, 401 N. Middletown Road, Pearl River, NY 10965. Phone: 845-602-3216; Fax: 845-602-5530; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-0939

�2016 American Association for Cancer Research.

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duration in some patients with AML who had achieved completebone marrow remission. For example, persistent thrombocytope-nia (<100,000/mL)was seen in13%ofpatients in a phase II clinicaltrial with amedian time of 66 days to recovery of 50,000 platelets/mL (10). Thrombocytopeniawas alsooneof themain side effectsofinotuzumab ozogamicin in clinical trials (grade 3/4 thrombocy-topenia in �30% of patients) and was one of the most commonreasons for dose delay, dose reduction, or treatment discontinu-ation (11). Liver effects reported with gemtuzumab ozogamicinand inotuzumab ozogamicin include increases in aminotrans-ferases, hyperbilirubinemia, and hepatic SOS. SOS, previouslyknown as hepatic veno-occlusive disease, is a serious medicalcondition characterized clinically by jaundice, painful hepatomeg-aly, weight gain, and ascites. SOS is a known complication ofhematopoietic stem cell transplantation (HSCT) as a result of theconditioning myeloablative regimen (12). In phase II trials ofgemtuzumab ozogamicin as a single agent at 9 mg/m2/dose fortwo doses separated by 2 weeks in first-relapse AML patients, 9%,18%, and 29% of patients experienced grade 3 or 4 alanineaminotransferase (ALT), aspartate aminotransferase (AST), andbilirubin elevations, respectively, and SOS was diagnosed in0.9% of patients in the absence of associated HSCT. In patientswho underwent HSCT prior to or after gemtuzumab ozogamicintreatment, the SOS rates rose to19%and15%, respectively (13). Ina phase II trial of inotuzumabozogamicin as a single agent given ata total dose of 1.8 mg/m2/cycle at 4-week intervals for up to sixcycles in patients with refractory or relapsed ALL, grade�3 hepaticaminotransferase elevations occurred in 6% of patients and SOSwas diagnosed in 9% of patients, with 67% of cases observed afterpost-study HSCT (14).

Similar thrombocytopenia and liver effects were therefore seenin patients with two calicheamicin conjugates targeting unrelatedantigens and are likely target-independent. Various mechanismshavebeenpostulated for these toxicities, includingmyelosuppres-

sion for platelet effects (1) and target expression onhepatocytes orKupffer cells for gemtuzumabozogamicin liver effects (15, 16). Asthe specific mechanisms are incompletely understood, an inves-tigative studywas performed in cynomolgusmonkeyswith a non-binding ADC containing the same linker and payload as gemtu-zumab ozogamicin and inotuzumab ozogamicin with the objec-tives to further investigate the mechanism for thrombocytopeniadevelopment, characterize the liver injury, and identify potentialbiomarkers [hyaluronic acid (HA), plasminogen activator inhib-itor-1 (PAI-1) and protein C] for the liver toxicity.

Materials and MethodsTest article

PF-0259 is an ADC that consists of a nonbinding human IgG1mAb linked to the cytotoxic agent N-acetyl gamma-calicheamicindimethyl hydrazide via the acid-labile 4-(40-acetylphenoxy) buta-noic acid linker. PF-0259 has the same linker and payload asgemtuzumab ozogamicin and inotuzumab ozogamicin. Charac-terization of PF-0259 was performed by size exclusion chroma-tography (Acquity UPLC BEH200 SEC, Waters), reverse-phaseultra-performance liquid chromatography (Zorbax 300SB-CN,Agilent) and hydrophobic interaction chromatography (TSKgelButyl-NPR, Tosoh Bioscience). The mAb did not bind specificallyto any antigen in protein array assays, polyreactivity assays usingDNA, insulin, and baculovirus particles and showed no bindingby IHC to cynomolgus monkey heart, kidney, liver, lung, skeletalmuscle, and spleen tissues. The average drug-to-antibody ratio ofPF-0259 was 3.9. PF-0259 showed cytotoxic effects comparablewith gemtuzumab ozogamicin and inotuzumab ozogamicin innon-target–expressing cell assays (e.g., average IC50 of 3.0 mg/mLfor PF-0259 and gemtuzumab ozogamicin in CD33-negative Rajicells). The test article PF-0259 was formulated in 20mmol/L Tris,8.5% sucrose, 0.02% polysorbate 80, and 0.005% disodiumEDTA (pH: 8.0), diluted in PBS, to achieve a final productconcentration of 0.5 mg/mL. The vehicle control article consistedof the formulation buffer diluted in PBS.

AnimalsMale cynomolgus monkeys (Macaca fascicularis) of Mauritius

origin, 3 to 4.5 years of age and weighing 3.5 to 6.3 kg at dosinginitiation, were obtained from a commercial supplier. Allprocedures performed on animals in this study were conductedin accordance with established guidelines and regulations, andwere reviewed and approved by Pfizer's Institutional AnimalCare and Use Committee. Pfizer animal care facilities thatsupported this work are fully accredited by the Association forAssessment and Accreditation of Laboratory Animal CareInternational.

Experimental designMale cynomolgus monkeys received up to 3 intravenous

bolus administrations 3 weeks apart on days 1, 22, and 43 ofthe vehicle control article (N ¼ 7) or PF-0259 at 6 mg/m2/dose(N ¼ 8) and were necropsied according to protocol random-ization on day 3 (i.e., 48 hours after the first administration—1vehicle control and 2 PF-0259–dosed monkeys) or on day 63(i.e., 21 days after the third administration—2 vehicle controland 6 PF-0259–dosed monkeys). A treatment cycle was definedas a single dose followed by a 3-week observation period.The test article PF-0259 and the vehicle control article were

Translational Relevance

Gemtuzumab ozogamicin (Mylotarg) and inotuzumabozogamicin are antibody–calicheamicin conjugates devel-oped for treatment of acute myeloid leukemia and acutelymphoblastic leukemia, respectively. Clinical adverse reac-tions with these drugs include thrombocytopenia andliver toxicity, characterized by increases in serum aminotrans-ferases and bilirubin with occasional cases of hepatic sinusoi-dal obstruction syndrome. This experimental work in cyno-molgus monkeys demonstrates a relationship between plate-let and liver effects, through an initial damage to liver sinu-soidal endothelial cells that is associated with plateletsequestration in liver sinusoids. This toxicity mechanism mayalso operate for other non-calicheamicin antibody–drug con-jugates (ADCs) with similar adverse events of thrombocyto-penia and liver microvascular injury. Evaluation of liver tox-icity biomarkers suggested that serum hyaluronic acidmay be a sensitive mechanism-based diagnostic marker. Thisreport may have implications for patient care as it providesbasis for research into new monitoring strategies, preventionapproaches, and treatment options through better under-standing of ADC toxicities.

Liver and Platelet Effects of Calicheamicin Conjugates

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administered at a dose volume of 1 mL/kg and the duration ofdosing was approximately 1 to 2 minutes. Four of the 7monkeys in the vehicle control group were used only forcollection of nonterminal parameters, were not necropsied,and were returned to the laboratory animal colony at comple-tion of the in-life phase. All animals were evaluated for clinicalsigns, changes in bodyweight, food consumption, clinical pathol-ogy parameters [including platelet counts, thrombopoietin andIL6), platelet activation inwhole blood, livermicrovascular injurybiomarkers (HA, PAI-1, and proteinC) and trough concentrationsof PF-0259 in serum as further described in the information tofollow. Light microscopy, transmission electron microscopy(TEM), histochemistry, and/or IHC were performed on selectedorgans of animals necropsied during the study as detailed below.

Experimental proceduresBlood sampling. Blood was collected for hematology in EDTAtubes, for clinical chemistry (including thrombopoietin, IL6 andHA) in serum separator tubes, and for coagulation (including PAI-1 and protein C) in sodium citrate tubes. All parameters wereevaluated predose (one or two time points) and on days 4, 11, 18,25, 32, 42, 46, 53, and 63. Hematology parameters and throm-bopoietinwere also evaluatedondays 2, 3, and7and IL6,HA, andPAI-1 on days 2 and 7.

Standard clinical pathology, thrombopoietin, and IL6. Standardhematology parameters were measured using a Siemens Advia2120 Hematology Analyzer and clinical chemistry parametersusing a Siemens Advia 1800 Chemistry Analyzer (SiemensHealthcare Diagnostics). Thrombopoietin and IL6 were assayedin serum samples by sandwich immunoassays using electroche-miluminescence and flow-based detection, respectively. Throm-bopoietin wasmeasured usingMSDHuman TPOKit (Meso ScaleDiscovery) and IL6 was assayed using Milliplex Non-HumanPrimate Cytokine Magnetic Bead Panel (EMD Millipore, MerckKGaA).

Liver microvascular injury biomarkers (HA, PAI-1, and protein C).HAwas assayed in serum samples using a specific sandwich ELISAkit (Echelon Biosciences Inc.). Citrated plasma was used for themeasurement of PAI-1 and protein C. Total PAI-1 concentrationswere determined with the Human Serpin E1/PAI-1 QuantikineELISA Kit (R&D Systems). Protein C activity was measured by aclotting assay using a STA Compact Hemostasis Analyzer (Diag-nostica Stago Inc.). Briefly, monkey plasma samples were firstincubated with a purified extract of Agkistrodon c. contortrixvenom for specific activation of protein C, which leads to inhi-bitionof coagulation factors V andVIII. ProteinCactivitywas thenassessed throughmeasurement of APTT in control human plasmasamples in which all coagulation factors were present, exceptprotein C, which was derived from the monkey samples beingtested.

Platelet activation inwhole blood.Platelet activationwas evaluatedpredose andondays 2 and18.Whole bloodwas collected fromallanimals into CTAD tubes (0.11 mol/L sodium citrate with the-ophylline, adenosine, and dipyridamole), which are specificallydesigned to prevent platelet activation after blood collection.Platelet activation was assessed by analysis of expression of CD61(Glycoprotein IIIa for identification of platelets) and CD62P(P-selectin for assessment of activation) by flow cytometry (FACS-Canto, Becton Dickinson). Positive control samples for plateletactivation were generated by adding adenosine diphosphate(ADP) to citrated blood samples from vehicle control animals.

Clinical pathology data evaluation. Individual animal data fromthe dosing period were compared with respective earliest baselinedata, unless otherwise specified. In addition, statistical analysisperformed for each parameter at each scheduled sampling timewas based on comparisons with concurrent vehicle controls. Eachparameter was analyzed either parametrically or nonparametri-cally using a two-sample test to compare groups. Testing for eachparameter was either one-sided for higher values, one-sided forlower values, or two-sided. These choices were prespecified foreach parameter. Statistically significant results were reported inthe graphs at the nominal 5%, 1%, 0.5%, or 0.1% level asappropriate.

Bioanalytical determinations. Residual serum samples from bloodcollected from PF-0259–treated monkeys at the end of each cycle(days 22, 43, and 63) were analyzed for PF-0259 and totalantibody concentrations.

Histopathology and histochemistry. Animals were fasted overnightbefore scheduled necropsies on days 3 and 63. Samples of liver,lung, kidney, heart, bone marrow (sternum), and spleen werefixed in 10% neutral buffered formalin, processed to paraffinblocks, and stained with hematoxylin and eosin (H&E) formicroscopic examination. In addition, silver stain for reticulinfibers and Masson trichrome stain for collagen fibers were per-formed on liver samples from animals necropsied on day 63.Bone marrow samples were obtained from flushed femurs andbone marrow smears were stained with May–Grunwald Giemsa.

IHC. VEGFR2 and CD31 IHC for endothelial cells, CD34 IHC forliver sinusoidal capillarization (17), CD68 IHC for monocytes/macrophages, and CD41 IHC for platelets were performed onsamples from all necropsied monkeys as detailed in Table 1. Allprimary antibodies used for IHCweremAbs,with the exception ofanti-CD41 polyclonal antibody.

Unstained 5-mm tissue sections were pretreated for antigenretrieval by heating slides in EDTA for CD31 and CD41 IHC orin Borg (Tris-based formulation, pH 9.5 - Biocare Medical) forCD34 IHC using a Decloaking Chamber. Slides were then loadedonto the Biocare intelliPATH for the staining protocol. Following

Table 1. Immunohistochemical stains performed on monkey tissue samples

Samples evaluatedMolecular target Antibody clone Day 3 Day 63

VEGFR2 55B11 (Cell Signaling Technology) Liver, kidney, lung LiverCD31 EP3095 (Abcam) Liver —

CD34 EP373Y (Abcam) — LiverCD68 514H12 (Leica Biosystems) Liver LiverCD41 Polyclonal (Sigma-Aldrich) Liver, kidney, lung, spleen Liver, spleen

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nonspecific blocking steps, the primary anti-CD31, -CD34, and-CD41 antibodieswere applied at dilutions of 1:200, 1:2,500, and1:500, respectively, for 1hour at room temperature.Detectionwasperformed using Dako EnVisionþ System with HRP polymer foramplification and Liquid DABþ for visualization (Dako). ForCD68 and VEGFR2 IHC, slides were loaded onto the Leica BondIII and pretreated with Epitope Retrieval Solution 2 (Leica Bio-systems). The anti-CD68 and the anti-VEGFR2 antibody wereapplied undiluted or at a dilution of 1:100, respectively, for 15minutes. Detection was accomplished by using Leica's RefinePolymer Kit. Isotype control rabbit or mouse IgGs were run onmatching tissue sections using similar protocols.

TEM. Liver samples from animals necropsied on day 3 wereprocessed for TEM. Samples were fixed in 2.5% glutaraldehydeand 2% paraformaldehyde in 0.1 mol/L phosphate buffer

(Karnovsky solution). After postfixation in 1% osmium tetrox-ide, samples were dehydrated through graded alcohols andpropylene oxide prior to embedding in resin. Thick sectionswere stained with toluidine blue for light microscopy detectionof areas of interest. Thin sections of areas of interest were placedon uncoated copper–palladium grids and stained with 10%lead citrate and 2.5% uranyl acetate.

ResultsPF-0259 induces acute thrombocytopenia in monkeys

The general physical condition of all monkeys was normalthroughout the experiment and there were no PF-0259–relatedeffects on body weight or food consumption.

Major PF-0259–related hematology changes consisted ofdecreases in platelet counts (Fig. 1A), with a consistent pattern

Figure 1.

PF-0259 induced thrombocytopenia in monkeys in the absence of myelosuppression or of direct effects on platelets in systemic circulation. Monkeys weredosed intravenously with vehicle or PF-0259 at 6 mg/m2/dose once every 3 weeks on days 1, 22, and 43. A, Effects of PF-0259 on blood platelet counts. PF-0259induced acute reversible thrombocytopenia during the first cycle and less pronounced but prolonged decreases in platelet counts during subsequent cycles.B, Evaluation of serum thrombopoietin concentration did not demonstrate upregulation of this hormone in association with thrombocytopenia. C, Histology ofsternal bone marrow on day 3 and day 63. There were no PF-0259–related alterations in hematopoietic cellularity and megakaryocyte density and morphology atboth necropsy time points. H&E stain, scale bar ¼ 60 mm. D, Evaluation of platelet activation in whole blood by flow cytometry analysis of expressionof CD61 (glycoprotein IIb for identification of platelets) and CD62P (P-selectin for evaluation of activation). Positive control samples were generated by addingadenosine diphosphate (ADP) to blood samples fromvehicle control animals. PF-0259 did not induce platelet activation in systemic circulation on days 2 and 18. Datain A, B, and D are represented as the mean values � 1 SD. Significantly different from vehicle control: � , P � 0.05; †, P � 0.01; z, P � 0.005; x, P � 0.001.






of acute reversible thrombocytopenia during the first cycle andless pronounced but prolonged reductions in platelet countsduring subsequent cycles. During the first cycle, decreases inplatelet counts were observed in all PF-0259–dosed monkeysstarting 24 hours (day 2) after PF-0259 administration (max0.53� baseline) with nadirs reached at 48 to 72 hours (0.14–0.64� baseline) and complete recovery by the end of the cycle.Subsequent PF-0259 administrations led to milder acute plateletreductions (e.g., 0.65–0.75� baseline in 4/6 monkeys 72 hoursafter the second administration) that did not recover to baselinevalues over the cycle durations with overall slow downward drifts(0.46–0.80� baseline at the end of the third cycle). Serumthrombopoietin was assessed to further characterize the throm-bocytopenia. There were decreases in circulating levels of throm-bopoietin in all PF-0259–dosed monkeys, mainly during the firstcycle from days 3 to 7 (down to 0.33� baseline) with lesspronounced effects seen during subsequent cycles in most ani-mals (Fig. 1B).

Additional PF-0259–related hematology changes of lesserimportance consisted of minimal decreases in red blood cellmass from day 7 and mild to moderate decreases in lymphocytecounts at various time points during the study (data notshown); the reductions in lymphocyte counts were associatedwith decreased lymphocyte cellularity in the spleen. There wereno PF-0259–related effects on other leukocyte populations,including neutrophils.

Trough concentrations of PF-0259 and total antibody in serumat the end of the cycles were overall similar to those observed for astructurally related ADC (data not shown), indicating adequatesystemic exposure and absence of antidrug antibodies.

The acute thrombocytopenia induced by PF-0259 is not due todecreased platelet production

Bone marrow assessment included histologic examination ofsternal bone marrow and cytologic evaluation of femoral bonemarrow cytospin smears. There were no PF-0259–related bonemarrow histologic or cytologic alterations in PF-0259–dosedmonkeys necropsied on day 3 or 63, and in particular, the overallhematopoietic cellularity and the megakaryocyte density andmorphology were within normal ranges at both necropsy timepoints (Fig. 1C).

In addition, considering the normal platelet lifespan of 6 to 8days in monkeys (18), the rapid decreases in platelet counts(down to 0.53� baseline 24 hours after PF-0259 administration)are consistent with peripheral destruction or sequestration ofplatelets rather than decreased production by the bone marrow.

PF-0259 induces acute liver endothelial cell toxicity associatedwith platelet sequestration in liver sinusoids

To investigate the potential mechanism for the acute throm-bocytopenia, selected monkeys were necropsied on day 3 at theapproximate platelet count nadirs.

Microscopic findings on day 3—liver. Although there were noremarkable PF-0259–related liver changes at light microscopicexamination of H&E-stained slides from monkeys necropsied onday 3 (Fig. 2A and B), IHC for endothelial cells and plateletsdemonstrated significant liver alterations in PF-0259–dosedmonkeys. As compared with controls, VEGFR2 IHC for endothe-lial cells showedmoderate tomarked loss of staining inmidzonaland, to a lesser extent, centrilobular regions (Fig. 2C and D),

which was corroborated by CD31 IHC for endothelial cells (datanot shown) andwas consistentwith loss of sinusoidal endothelialcells (SEC). In addition, CD41 (glycoprotein IIb) IHC for plateletsshowedmoderate tomarked granular staining in sinusoids,whichwas indicative of platelet accumulation, in midzonal regionsthroughout the sections in PF-0259–dosed monkeys (Fig. 2E–H). There were no appreciable differences in CD68 immunostain-ing for monocytes/macrophages (including Kupffer cells)between vehicle control and PF-0259–dosed monkeys (data notshown). TEM evaluation of liver samples similarly indicateddegeneration and loss of SECs associated with accumulation ofnumerous platelets in sinusoids, predominantly in midzonalregions (Fig. 3). In addition, sequestered platelets frequentlyexhibited alterations in cell shape (with formation of surfaceprojections and filopodia in particular) and reduced number ordensity of cytoplasmic granules, consistent with platelet activa-tion and degranulation, respectively (19). Sinusoids in affectedareas often contained sloughed necrotic cells (likely endothelialcells) and scattered leukocytes.

Microscopicfindings on day 3—other organs. To investigate the liverspecificity of the findings reported previously, VEGFR2 andCD41 IHC were performed on kidney and lung samples frommonkeys necropsied on day 3 and confirmed the absence ofsimilar changes in these two organs (CD41 IHC in SupplementaryFig. S1). CD41 IHC for platelets in spleen showed reducedstaining in PF-0259–dosed monkeys as compared with the con-trolmonkey, indicating release of platelets from the spleen storagepool secondary to PF-0259–related systemic thrombocytopenia(Supplementary Fig. S2A and S2B).

Platelet activation and cytokine release in systemic circulation. Toconfirm the lack of direct effects on platelets in the systemiccirculation, platelet activation in whole blood and cytokinerelease (IL6) in serum were assessed. There were no PF-0259–related increases in activated platelet counts, as assessed by flowcytometry evaluation of platelet CD62P (P-selectin) expression,on days 2 and 18 in all monkeys when compared with respectiveindividual baseline data. The ADP-stimulated positive controlsamples showed 33–165� increases in activated platelets whencompared with unstimulated control samples, demonstratingvalidity of the assay (Fig. 1D). There were no PF-0259–relatedchanges in IL6 throughout the study (Supplementary Fig. S3).

In summary, day 3 investigations demonstrated PF-0259–mediated liver-specific endothelial injury associated with plateletsequestration in sinusoids, which was considered responsible forthe acute thrombocytopenia.

Liver endothelial damage may progress to early SOS changesTo characterize the evolution of liver endothelial damage,

microscopic liver findings were characterized in animals necrop-sied on day 63 (21 days after the third administration).

PF-0259–related liver findings at light microscopic examina-tion of H&E-stained slides were generally minimal to mild andconsisted mainly of multifocal, usually midzonal, sinusoidaldilation and/or hepatocyte atrophy that were best appreciatedwith a reticulin stain. Sinusoidal dilation was characterized by thepresence of small irregular foci of variably dilated sinusoids oftenfilled with red blood cells and separated by thin cords of atrophichepatocytes. Small foci of hepatocyte atrophy were also observedin the absence of sinusoidal dilation. Silver stain for reticulin

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fibers further highlighted the finding through the delineation offocally thin hepatocellular plates (Fig. 4A and B). In addition,in areas of sinusoidal dilation or hepatocyte atrophy, Massontrichrome stain for collagen fibers showed delicate sinusoidalfibrosis. Other microscopic findings included minimal, multi-focal hepatocyte hypertrophy/regeneration and minimal pig-ment deposition in Kupffer cells in a single PF-0259–dosedmonkey.

VEGFR2 and CD34 IHC were performed on liver samples tofurther demonstrate and characterize endothelial cells. As com-paredwith day 3when there wasmarked loss of VEGFR2 staining,VEGFR2 IHC on day 63 samples showed only minor and focalalterations in endothelial cell staining in PF-0259–dosed mon-keys, mostly in areas of hepatocyte atrophy (Fig. 4C and D),indicating significant recovery of the initial endothelial damage.While CD34 IHC in control monkeys only stained endothelialcells associated with or in close proximity of the portal triads andcentral veins (Fig. 4G), there was mild to marked CD34 immu-nostaining of SECs throughout the liver lobules in PF-0259–dosed monkeys (Fig. 4H). CD34 overexpression is a marker ofsinusoidal capillarization and has been associated with liverfunctional impairment (17).

These microscopic liver findings in monkeys, including inparticular sinusoidal capillarization and sinusoidal dilation/hepatocyte atrophy, were qualitatively similar to those reported

in humans after administration of oxaliplatin and were consid-ered to represent early subclinical stages of SOS (17, 20).

Finally, there were no appreciable differences in CD41 immu-nostaining for platelets (Fig. 4E and F) and CD68 immunostain-ing for monocytes/macrophages (including Kupffer cells) in liversamples between control and PF-0259–dosed monkeys on day63. Similarly, there were no appreciable differences in CD41immunostaining for platelets in spleen samples between controland PF-0259–dosed monkeys on that day (Supplementary Fig.S2C and S2D).

Hyaluronic acid may be a sensitive mechanism-baseddiagnostic marker of PF-0259–related liver injuryStandard clinical chemistry and coagulation. PF-0259–relatedchanges in standard liver parameters were limited to overallminimal increases in AST levels (up to 3.09� baseline), withonset usually on day 4 and sustained effects over the studyduration (Fig. 5A). Changes in ALT levels in PF-0259–dosedmonkeys were inconsistent. In addition, there were minimal tomild PF-0259–related increases in activated partial thromboplas-tin time (APTT, up to 1.33�baseline), whichwere notedmostly at72 hours after each administration with APTT peaks seen after thefirst dose. Increases infibrinogen (up to 2.04� baseline), globulin(up to 1.19� baseline) and decreases in albumin (down to 0.84�baseline) were observed in some PF-0259–dosed monkeys at

Figure 2.

PF-0259 induced acute liver sinusoidalendothelial cell injury associated withintrasinusoidal platelet sequestration.Monkeys were necropsied 48 hoursafter a single intravenousadministration of vehicle or PF-0259 at6 mg/m2. Light microscopic evaluationof liver from vehicle control (A, C, E, G)and PF-0259–dosed (B, D, F, H)monkeys. There were no remarkablePF-0259-related liver changes at lightmicroscopic examination of H&E-stained slides (A and B). VEGFR2 IHCshowed delicate and diffuse staining ofsinusoidal endothelial lining cells incontrol monkey (C) while there wasstaining disruption and marked loss ofVEGFR2 immunoreactivity in midzonaland to a lesser extent centrilobularregions in PF-0259–dosed monkey (D).CD41 IHC for platelets showed minimalscattered punctate staining in vascularspaces in control monkey (E and G),while there was abundantintrasinusoidal granular staining inmidzonal regions throughout the liversections in PF-0259–dosed monkey,consistent with platelet accumulation(F, H). The lower magnification (F)demonstrated the midzonaldistribution and the highermagnification the intrasinusoidallocation (H) of the staining. pa,portal area; cv, central vein. Scalebar ¼ 100 mm.






various time points and possibly related to an inflammatoryresponse.

Although AST and APTT increases are likely related to the liverinjury, these parameters are not considered suitable diagnosticmarkers of the liver condition due to the poor liver specificity,narrow dynamic range in this study and/or uncertain mechanismfor the changes.

Exploratory biomarkers. Following a review of the available liter-ature, potential biomarkers of liver endothelial cell injury mea-sured included PAI-1, protein C, and HA (21, 22). There were noPF-0259–related changes in PAI-1 concentration (Fig. 5C) andprotein C activity (Fig. 5D) throughout the study. Biomarkerchanges were limited to increases in serum HA concentration inall PF-0259–dosed monkeys (Fig. 5B). HA is a polysaccharidepresent in the extracellular matrix that is synthesized by mesen-chymal cells throughout the body and cleared, almost exclusively,by liver SECs (23). Although some fluctuation in serum HA levelwas seen in control monkeys over the study duration, there wereonly 2 individual values in control monkeys slightly above 100ng/mL (both in the same animal). Therefore, individual values inPF-0259–dosed monkeys >100 ng/mL with increases >2-fold thehighest respective baseline values were considered to represent aneffect of PF-0259. Increases in serum HA concentrations wereobserved from day 2 (up to 4.71� baseline), peaked on day 4 (upto 6.76�baseline) with a trend for recovery over the first cycle andsustained increases over subsequent cycles (up to8.49�baseline).Increases in HA concentrations correlated well with AST levels(R¼ 0.84) and with liver microscopic findings of SEC loss on day3 and sinusoidal capillarization and/or sinusoidal dilation/hepa-tocyte atrophy on day 63. In particular, the highest HA value(311.9 ng/mL) on day 63 was recorded in a monkey who had thehighest AST increase (3.09� baseline) and liver pathology. The

data from this study suggest that elevated serum HA levels inmonkeys may be due to decreased clearance by injured hepaticSECs and thatHAmay be a sensitivemechanism-based diagnosticmarker of liver microvascular injury.

DiscussionThrombocytopenia and hepatic injury including SOS aremajor

adverse events reported in some patients treated with antibody–calicheamicin conjugates such as gemtuzumab ozogamicin andinotuzumab ozogamicin. SOS is a potentially life-threateningliver disorder that is thought to be initiated by injury to SECs(24). We showed that thrombocytopenia and microscopic liverinjury consistent with early SOS were similarly seen in monkeysdosed with PF-0259, a nonbinding ADC containing the samelinker and payload as gemtuzumab ozogamicin and inotuzumabozogamicin.

Themechanism for thrombocytopenia developmentwas inves-tigated in monkeys. The three main mechanisms for a reducedplatelet count are impaired production, increased destruction,and/or altered distribution (e.g., splenic sequestration; ref. 25).The current study suggests that the acute thrombocytopeniaobserved in PF-0259–dosed monkeys was not due to decreasedproduction of platelets by the bone marrow but resulted fromliver sequestration of platelets following liver-specific endothelialinjury. The rapid decreases in platelet counts, with onset at 24hours and up to 86% reduction at 72 hours after PF-0259administration, were not consistent with an effect on bone mar-row production, given the normal platelet lifespan of 6 to 8 daysin monkeys (18). Marked platelet accumulation in sinusoidssecondary to SEC degeneration and loss was demonstrated byIHC and TEM in liver samples frommonkeys necropsied 48 hoursafter PF-0259 administration. Similar changes were not present in

Figure 3.

Electron microscopic (EM) evaluation of liver samples confirmed PF-0259–related acute midzonal sinusoidal endothelial cell (SEC) injury associated withplatelet sequestration. Monkeyswere necropsied 48 hours after a single intravenous administration of vehicle or PF-0259 at 6mg/m2. Transmission EM evaluation ofliver from vehicle control (A) and PF-0259–dosed (B) monkeys. A, Note the normal aspect of the fenestrated endothelium (arrows) lining the space ofDisse in the control monkey. � , Lumen of sinusoid; H, hepatocyte; RBC, red blood cell. B, There was loss of sinusoidal endothelial lining cells with direct contactbetween the space ofDisse and the lumen of the sinusoids (black arrows) in the PF-0259–dosedmonkey. The endothelial cell losswas associatedwith intrasinusoidalaccumulation of platelets (red arrows) showing alterations in cell shapes such as surface projections. There were also scattered sloughed necrotic cells(likely endothelial cells) and leukocytes in the lumen of the sinusoids. Scale bar ¼ 1 mm.

Guffroy et al.





the lung andkidney from thesemonkeys,whichdemonstrated theliver specificity of these alterations.

Our data are consistent with recently published investigationson the mechanism of SOS-associated thrombocytopenia in otherclinical settings (26–28). Liver samples from patients with SOSfollowing oxaliplatin-based chemotherapy or liver transplanta-tion were immunostained for platelet surface marker CD42b(glycoprotein Iba) or CD41 (glycoprotein IIb) and demonstratedplatelet aggregates or "microthrombi" in sinusoids usually incentrilobular regions. Investigators concluded that plateletsequestration in the liver following SEC damage was the likelymechanism for the observed thrombocytopenias. In addition, therapid consumption of transfused platelets that is frequentlyobserved in SOS patients (29, 30) is likely due to the samemechanism.

Although liver SEC damage is the initiating event, plateletaccumulation andactivationwithinhepatic sinusoidsmay furtherexacerbate the liver injury through release of potentially damagingplatelet granule contents. Pharmacologic inhibition of plateletactivation/degranulation might therefore mitigate hepatic injuryand SOS in patients. The toxicity of activated platelets to endo-thelial cells has been shown in vitro and, among the substancesreleased by platelets, thromboxane A2 and serotonin were shownto cause endothelial cell damage while PDGF had no effects (31).Nakanuma, Nishigori, and Tajima similarly hypothesized that

release of various growth factors by activated platelets (such asthromboxane A2, thrombospondin and VEGF-A) might contrib-ute to the progression of SOS in patients (26–28). Studies haveshown that the anti-VEGF-A antibody, bevacizumab, protectsagainst oxaliplatin-associated SOS (20, 21, 32) and the mecha-nism, although not completely understood, could involve inhi-bition of VEGF-A released from activated platelets. Recently,cilostazol, a phosphodiesterase 3 inhibitor with antiplatelet prop-erties, was reported to prevent SOS following living donor livertransplantation (33).

Although the mechanism for the acute thrombocytopenia inmonkeys has been elucidated, the cause for the lack of completeplatelet recovery following repeated administrations of PF-0259remained incompletely understood, given the absence of bonemarrow damage or of significant platelet sequestration in the liveron day 63. As thrombopoietin is mainly produced by the liver(34), the observation of decreases in serum thrombopoietinmight be related to the ongoing liver alterations and indicativeof inadequate bone marrow stimulation with concomitantdecreased ability to achieve normal platelet numbers.

Microscopic liver findings in monkeys after repeated adminis-trations of PF-0259 included sinusoidal capillarization and sinu-soidal dilation/hepatocyte atrophy, which have been described inpatients with SOS (17, 20). As compared with day 3, there wassignificant recovery of the initial endothelial cell loss with CD34

Figure 4.

Liver sinusoidal endothelial damageprogressed to microscopic changesconsistent with early stages of sinusoidalobstruction syndrome (SOS). Monkeyswere dosed intravenously with vehicle orPF-0259 at 6 mg/m2/dose once every 3weeks and were necropsied on day 63 atthe end of the third cycle. Lightmicroscopic evaluation of liver fromvehicle control (A, C, E, G) and PF-0259-dosed (B, D, F, H) monkeys. The reticulinstain showed regularly sizedhepatocellular plates in the controlmonkey (A) while it clearly demonstratedscattered, usually midzonal, foci ofhepatocellular atrophy in PF-0259–dosedmonkey (B). VEGFR2 IHC for endothelialcells (C and D) showed focal alterationand loss of VEGFR2 staining in areas ofhepatocellular atrophy in PF-0259–dosedmonkey (D), consistent with focalpersistent loss of sinusoidal endothelialcells (SEC). CD41 IHC for platelets (E andF) did not demonstrate any significantsequestrationofplatelets in liver sinusoidsin PF-0259–dosed monkey at this stage(F). CD34 IHC only stained endothelialcells associated with or in close proximityof the portal triads and central veins in thecontrol monkey (G) while there wasmoderate to marked CD34immunostaining of SECs throughout theliver lobules (outside of areas ofhepatocellular atrophy) in the PF-0259–dosed monkey, which is indicative ofsinusoidal capillarization (H). pa, portalarea; cv, central vein. Scale bar¼ 100 mm.






overexpression by recovered cells. CD34 expression is amarker ofsinusoidal capillarization, which is a pathologic finding charac-terized by alterations of endothelial cells (i.e., development of abasement membrane and loss of fenestrations) and liver func-tional impairment (17, 35). In addition, the observation of CD34overexpression suggests recovery of endothelial damage throughrecruitment of bone marrow–derived CD34-positive endothelialprogenitor cells, as has been shown in other vascular injurysettings (36). Recently, infusion of endothelial progenitor cellswas shown to mitigate liver injury in mice after HSCT (37).

Another objective of our work was to evaluate biomarkers ofliver injury. There are currently no validated blood tests for SOSand the diagnosis is mainly based on clinical and laboratorycriteria that are nonspecific and/or late events in the developmentof the disease (12). SOS-associated endothelial injury triggers ahypercoagulable state and several studies have evaluated inhuman patients the diagnostic value of proteins involved incoagulation, such as PAI-1 and protein C (22, 38), with incon-sistent results. PAI-1 is an inhibitor of fibrinolysis that is synthe-tized and released by endothelial cells and protein C has antico-agulant activity through inactivation of factors Va and VIIIa. Therewere no PF-0259–related changes in PAI-1 and protein C in any

monkey throughout the study and these parameters were notsensitive markers of endothelial damage in these experimentalconditions. HA is a polysaccharide present in the extracellularmatrix that is synthesized by mesenchymal cells and cleared,almost exclusively, by liver SECs (23). Systemic HA levels can beincreased with liver SEC structural and/or functional damage andhave been explored clinically for the noninvasive detectionof SOSwith promising results (21, 39). Evaluation of HA levels inmonkeys showed early and sustained increases in HA that corre-lated well with AST levels and liver pathology on both days 3 and63, indicating the ability of this parameter to detect both earlystructural damage (day 3) and later functional impairment (day63) of SECs. HA is, however, not a specific marker of liver SECdamage as there are other mechanisms that can lead to increasedserum levels, such as increased HA production during the courseof fibroplasia (40, 41). Despite the limitations related to the lackof HA specificity, our data provide additional support to thediagnostic value of HA for the sensitive detection of liver micro-vascular injury, which should be explored in future work forconfirming its potential as a biomarker in humans.

Finally, we hypothesize that the mechanisms demonstratedin monkeys for the target-independent liver injury and

Figure 5.

Effects of PF-0259 on potential circulating markers of liver injury. Monkeys were dosed intravenously with vehicle or PF-0259 at 6 mg/m2/dose once every3 weeks. There were early and sustained increases in hyaluronic acid (HA; up to 8.5�; B) that correlated well with AST levels (A) and liver pathology on both days3 and 63. There were no PF-0259–related changes in plasma plasminogen activator inhibitor-1 (PAI-1) concentration (C) and protein C activity (D) throughoutthe study. Data are represented as the mean values � 1 SD. Significantly different from vehicle control: � , P � 0.05; †, P � 0.01; z, P � 0.005; x, P � 0.001.

Guffroy et al.





thrombocytopenia of antibody–calicheamicin conjugates mayoperate for other classes of ADCs. Trastuzumab emtansine(T-DM1) is an ADC composed of a microtubule inhibitor con-jugated via a stable linker to a humanized mAb against HER2.Major adverse events in patients include thrombocytopenia andliver effects. The thrombocytopenia is usually characterized by: (i)acute onset with decreases in platelet counts as soon as 1 day afterT-DM1 administration (42), (ii) platelet nadirs during cycle 1 (3-week cycle) with recovery by end of cycle, and (iii) slow down-ward drifts in platelet counts in some patients over multiple T-DM1 cycles (43). In addition, it is noteworthy that hematologiceffects with T-DM1 are quite selective for platelets and otherhematologic lineages are relatively unaffected. Liver effects withT-DM1 are characterized by increases in serum aminotransferasesalong with occasional cases of nodular regenerative hyperplasia(NRH; ref. 44). NRH is a liver microvascular disorder that, similarto SOS, is thought to result from an initial insult to liver SECs.Interestingly, conditions associated with SOS development, suchas HSCT procedure and oxaliplatin treatment, are also associatedwith NRH development, suggesting a pathogenetic link betweenSOS and NRH. The overall similarity of effects on platelets andliver with T-DM1 and antibody–calicheamicin conjugates sug-gests that a similar underlying mechanism may contribute to thethrombocytopenia and liver injury with both classes of drugs.However, these similarities require further investigation in light ofanother recently proposed hypothesis for T-DM1–mediated liverinjury, namely HER2 expression by hepatocytes (45).

In conclusion, this work has investigated the liver toxicity andthrombocytopenia of antibody–calicheamicin conjugates andsuggested a relationship in monkeys between these two patho-logic processes, through target-independent damage to liver SECsthat is associatedwith platelet sequestration in liver sinusoids.Wefurther hypothesize that a similar mechanism may operate forother ADCs in patients where adverse events of thrombocytope-nia, increased liver enzymes, and liver microvascular disorders(including NRH) have been observed.

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

Authors' ContributionsConception and design: M. Guffroy, H. Falahatpisheh, L. Obert, H.-P. Gerber,M.B. Finkelstein, N.K. KhanDevelopment of methodology: M. Guffroy, J. Kreeger, L. Obert, T. Coskran,D. Sullivan, C. Huang, M.B. Finkelstein, N.K. KhanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Biddle, J. Kreeger, L. Obert, R. Goldstein,D. Sullivan, C. Huang, R. Giovanelli, N.K. KhanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):M.Guffroy, K. Biddle, J. Kreeger, L. Obert, W. Reagan,C. Huang, S.A. Sokolowski, N.K. KhanWriting, review, and/or revision of the manuscript: M. Guffroy, H. Falahat-pisheh, K. Biddle, J. Kreeger, L. Obert, K. Walters, R. Goldstein, W. Reagan,D. Sullivan, S.A. Sokolowski, H.-P. Gerber, M.B. Finkelstein, N.K. KhanAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K. Walters, G. Boucher, D. SullivanStudy supervision: M. Guffroy, H. Falahatpisheh, K. Walters, W. Reagan,N.K. Khan

AcknowledgmentsThe authors thankKiranKhandke for designing andproducing the antibody–

calicheamicin conjugate used in this study, Walter Bobrowski and GermaineBoucher for excellent assistance with the photomicrographs, and Sarah Lopesfor quality control of the manuscript.

Grant SupportThis work was supported by Pfizer Inc.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 14, 2016; revised August 17, 2016; accepted September 9,2016; published OnlineFirst September 28, 2016.

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Guffroy et al.




2017;23:1760-1770. Published OnlineFirst September 28, 2016.Clin Cancer Res Magali Guffroy, Hadi Falahatpisheh, Kathleen Biddle, et al. and Monitoring

Mechanism−−Calicheamicin Conjugates in Cynomolgus Monkeys−Liver Microvascular Injury and Thrombocytopenia of Antibody

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