Intracellular Activation of SGN-35, a Potent Anti-CD30 Antibody … · Purpose: SGN-35 is an...

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Published OnlineFirst January 19, 2010; DOI: 10.1158/1078-0432.CCR-09-2069 Published OnlineFirst January 19, 2010; DOI: 10.1158/1078-0432.CCR-09-2069 Published OnlineFirst January 19, 2010; DOI: 10.1158/1078-0432.CCR-09-2069

Cancer Therapy: Preclinical Clinical

Cancer

Research

Intracellular Activation of SGN-35, a Potent Anti-CD30Antibody-Drug Conjugate

Nicole M. Okeley, Jamie B. Miyamoto, Xinqun Zhang, Russell J. Sanderson, Dennis R. Benjamin,Eric L. Sievers, Peter D. Senter, and Stephen C. Alley

Abstract

Authors' A

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Purpose: SGN-35 is an antibody-drug conjugate (ADC) containing the potent antimitotic drug, mono-methylauristatin E (MMAE), linked to the anti-CD30 monoclonal antibody, cAC10. As previously shown,SGN-35 treatment regresses and cures established Hodgkin lymphoma and anaplastic large cell lympho-ma xenografts. Recently, the ADC has been shown to possess pronounced activity in clinical trials. Here,we investigate the molecular basis for the activities of SGN-35 by determining the extent of targetedintracellular drug release and retention, and bystander activities.Experimental Design: SGN-35 was prepared with 14C-labeled MMAE. Intracellular ADC activation on

CD30+ and negative cell lines was determined using a combination of radiometric and liquid chromato-grahpy/mass spectrometry-based assays. The bystander activity of SGN-35 was determined using mixedtumor cell cultures consisting of CD30+ and CD30− lines.Results: SGN-35 treatment of CD30+ cells leads to efficient intracellular release of chemically unmod-

ified MMAE, with intracellular concentrations of MMAE in the range of 500 nmol/L. This was due tospecific ADC binding, uptake, MMAE retention, and receptor recycling or resynthesis. MMAE accountsfor the total detectable released drug from CD30+ cells, and has a half-life of retention of 15 to 20 h.Cytotoxicity studies with mixtures of CD30+ and CD30− cell lines indicated that diffusible released MMAEfrom CD30+ cells was able to kill cocultivated CD30− cells.Conclusions: MMAE is efficiently released from SGN-35 within CD30+ cancer cells and, due to its

membrane permeability, is able to exert cytotoxic activity on bystander cells. This provides mechanisticinsight into the pronounced preclinical and clinical antitumor activities observed with SGN-35. Clin CancerRes; 16(3); 888–97. ©2010 AACR.

Several monoclonal antibodies (mAb) have establishedroles in cancer chemotherapy due to their specificities fortumor-associated antigens and their manageable off targettoxicities (1, 2). In almost all cases, these agents are usedin combination with chemotherapeutic regimens becausetheir activities as single agents are generally suboptimal(3). To extend and enhance this approach, cytotoxic drugshave been linked to mAbs, generating antibody-drug con-jugates (ADC) that are capable of selectively deliveringdrug to target sites. In doing so, it is possible to increasedrug activity and at the same time reduce toxic side effectsthrough selective delivery.Although the ADC concept has long been explored, gem-

tuzumab ozogamicin (Mylotarg) is the only one that hasbeen approved by the Food and Drug Administration. This

ffiliation: Seattle Genetics, Inc., Bothell, Washington

lementary data for this article are available at Clinical Cancernline (http://clincancerres.aacrjournals.org/).

ding Author: Nicole M. Okeley, Seattle Genetics, Inc., 21823Southeast, Bothell, WA 98021. Phone: 425-527-4748;7-4001; E-mail: [email protected].

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agent is composed of a mAb recognizing the CD33 receptoron acute myelogenous leukemia, modified with the highlypotent cytotoxic agent calicheamicin through a hydrolyti-cally unstable linker (4–6). In the years since the approvalof gemtuzumab ozogamicin, the progress in developingnewer generation ADCs for cancer therapy has been consid-erable, with increased understanding of the roles that thetarget antigen, drug potency, linker stability, and conju-gation methods play in ADC efficacy and tolerability(7–9). SGN-35 and trastuzumab-DM1 are two suchagents that address many of the key parameters, andboth have shown activities in phase I and II clinicaltrials (10–15).SGN-35 (Fig. 1) is directed against the CD30 antigen,

which is highly expressed on such tumors as Hodgkin lym-phoma (HL) and anaplastic large cell lymphoma. ThecAC10 mAb component of SGN-35 is empowered by theaddition of an average of four molecules of monomethy-lauristatin E (MMAE), a synthetic antimitotic agent thatpotently inhibits tubulin polymerization leading to apop-totic cell death (16–20). A dipeptide linker is used to attachMMAE to cAC10 in such away that upon in vitro exposure toproteolytic enzymes such as cathepsin B, free MMAE is re-leased (16, 21). This is expected tooccur inside the lysosomes

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Translational Relevance

SGN-35 is one of the few antibody drug conjugateswith significant clinical efficacy. The agent consistsof a potent cytotoxic drug monomethylauristatin E(MMAE), attached to an anti-CD30 monoclonalantibody through a cleavable dipeptide linker. SGN-35 quantitatively releases and accumulates MMAE in-side CD30+ cells, and this is accompanied by extendedMMAE cellular residence. Sustained contact with theactive drug through SGN-35 treatment is clinically rel-evant because extended exposure may not allow tumorcells a chance to escape the effects of MMAE. We alsoshow that effluxed MMAE can kill CD30− tumor cellscocultured with CD30+ cells, suggesting that this tar-geting technology is applicable to monoclonoal anti-bodies against heterogeneously expressed targets onhuman tumors. The molecular pharmacology of SGN-35 and the ability of the released drug to exert bystandereffects provide a framework for understanding the activ-ities of this agent in treating heterogeneous tumors,such as Hodgkin lymphoma.

Released Drug from SGN-35

Published OnlineFirst January 19, 2010; DOI: 10.1158/1078-0432.CCR-09-2069

of cells after the ADC is internalized through receptor-mediated endocytosis.The preclinical activities of SGN-35 are pronounced. At

the in vitro cellular level, the ADC kills CD30-positivecells at low pmole concentrations, several orders of mag-nitude lower than the amount required for antigen satu-ration (16, 17). In vivo studies have shown that SGN-35treatment leads to the regression and cure of establishedHL human tumor xenografts in mice at doses in therange of 1 mg/kg, which is far below the maximum tol-erated dose of 120 mg/kg (18). Based on these activities,we initiated a phase I clinical trial of single agent SGN-35 in patients with relapsed, refractory HL, and otherCD30-positive hematologic malignancies. Treatment withthis ADC has provided multiple objective responses, witha substantial proportion of the patients having completeresponses (10, 13, 15).The identity of the active chemical species released from

SGN-35 within cancer cells has not been reported. This is-sue is of importance to gain further insight into the molec-ular basis for SGN-35 activity and its application fortreating clonal malignant populations that heterogeneous-ly express targeted cell surface antigens, in addition to thefact that mAbs have been shown to distribute within tu-mors in an uneven manner (22). HL consists of CD30-positive Reed-Sternberg cells surrounded by polyclonal,reactive tumor-associated macrophages, fibroblasts, eosi-nophils, mast cells, B cells, plasma cells, and T cells thatare CD30 negative (23–25). Here, we describe studies thatidentify MMAE as the released drug within CD30-positiveHL cells treated with SGN-35. Intracellular concentrations

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of released drug are high over a prolonged time period, yetthe amount of effluxed drug is sufficient to exert bystanderactivity on cocultured antigen-negative cells. These studiesprovide mechanistic insight into the activities and proper-ties of SGN-35, and a basis for using this agent for thetreatment of tumor cell populations that express theCD30 antigen in a heterogeneous manner.

Materials and Methods

Radiolabeled drugs and ADCs. 14C-labeled MMAE andvc-MMAE were prepared through custom synthesis byPerkin-Elmer (14C-MMAE and 14C-vc-MMAE). MMAEand vc-MMAE were labeled at the second valine (univer-sally 14C-labeled, 270 mCi/mmol MMAE and 276 mCi/mmol vc-MMAE; Fig. 1). cAC10-14C-vc-MMAE was pre-pared with a method similar to that previously describedusing partial reduction by DTT (26). The remaining un-reacted DTT was removed by PD10 size exclusion chroma-tography (GE Healthcare) and excess 14C-vc-MMAE wasadded to conjugate at 0°C. Any unreacted drug linker wasquenched with cysteine and the conjugate was purifiedfrom unconjugated small molecule by PD10. The specificactivity of the ADC was determined by UV/visible spectros-copy and liquid scintillation counting (LSC). The specificactivity of the unconjugated drug linker was used to calcu-late ADC's drug loading (7.9 μCi/mg, 4.4 drugs/antibody).Cell culture. The CD30-positive cell line Karpas299 (an-

aplastic large cell lymphoma) and the CD30-negative lineWSU-NHL (non-Hodgkin lymphoma) were obtainedfrom the Deutsche Sammlung von Mikroorganism undZellkulturen GmbH. The CD30-negative line Ramos wasobtained from American Type Culture Collection. TheCD30-positive cell line L540cy, a derivative of the HL lineL540 adapted to xenograft growth, was provided by Dr.Philip Thorpe (University of Texas Southwestern MedicalCenter, Dallas, TX). Cells were grown in suspension cul-ture in RPMI 1640 supplemented with either 10% fetalbovine serum (FBS; Invitrogen, Carlsbad; Karpas299,WSU-NHL, and Ramos) or 20% FBS (L540cy) in a humid-ified environment of 5% CO2 at 37°C.Cell volume determination. Cell volumes were deter-

mined by estimation of the average viable cell diameterusing a Vi-Cell XR2.03 cell viability analyzer (BeckmanCoulter) that provides the average cell size in microns.Volume calculation assumed a spherical cell with no cor-rection made for nuclear volume.ADC catabolism and drug distribution (constant exposure).

Cells were seeded at 5 × 105 cells/mL. Cultures were exam-ined in triplicate and typical culture volumes were 30 mL.On each day of the 72-h experiment, cell densities and vi-abilities were determined by trypan blue exclusion. Radio-active SGN-35 was added to each culture at a finalconcentration of ∼200 ng/mL (6 nmol/L drug equivalent).Flasks were swirled to mix and aliquots were removedfrom each flask as a reference for the total amount of ra-dioactivity added to the culture. The cultures were incub-ated at 37°C in a humidified 5% CO2 atmosphere. At the

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indicated time points, cultures were mixed and 4 mL ali-quots were removed and overlaid on FBS cushions (27) in15-mL centrifuge tubes. The samples were centrifuged at390 × g for 5 min at room temperature. From the separatedsample, an aliquot of the upper medium phase was re-moved for further analysis (see below). The remaining super-natant was carefully removed. The pellet was resuspended in4 mL of ice-cold PBS and 1 mL was removed for LSC quan-titation of total cell-associated radioactivity. The remaining3 mL of cells were pelleted and resuspended in 0.5 mL ofproteinase K (5 μg/mL; Promega) in PBS to removesurface-bound ADC. After incubation at 37°C for 10 min,the enzyme was quenched by dilution with 1 mL of FBS-containing medium. The entire sample was overlaid onan FBS cushion (27) and centrifuged at 390 × g for 5 min.The pellet was resuspended in 100 μL of complete mediumand was then treated with 900 μL of ice-cold methanol toprecipitate protein and permeabolize the cells. An aliquotof this cell precipitation suspension (400 μL) was removedand examined by LSC to quantitate the total intracellularradioactivity. The remaining suspension was stored at−20°C for ≥30 min. The sample was then centrifuged at16,000 × g for 5 min. The clarified supernatant (500 μL)was examined by LSC to quantitate the total intracellularradioactive small molecule.A sample of the culture medium from each time point

was diluted with nine volumes of ice-cold methanol. Theresulting suspension was stored at −20°C for ≥30 min.The sample was then centrifuged at 16,000 × g for 5 minand the supernatant was counted by LSC for quantitationof the nonprotein-associated radioactivity present in theculture medium. All samples used for LSC were mixedwith 4 mL of Ecoscint A scintillation fluid and were vor-texed. L540cy cells treated with chloroquine (Sigma) were

Clin Cancer Res; 16(3) February 1, 2010


preincubated with 100 μmol/L chloroquine at 37°C for1 h followed by 5 h of incubation with radiolabeledSGN-35 and subsequent processing as described above.ADC catabolism and drug distribution (limited exposure).

To examine ADC catabolism and drug distribution in cellswith limited ADC exposure, cultures were prepared as de-scribed above using ice-cold culture medium and wereplaced on ice before ADC addition. After incubation onice for ∼15 min, 14C-SGN-35 was added at ∼200 ng/mLand the cultures were kept on ice for an additional 30 min.Cultures were then centrifuged at 390 × g for 5 min andwere washed with ice-cold PBS followed by resuspensionin fresh, warm culture medium. The cultures were placedat 37°C in a humidified 5% CO2 atmosphere and sampleswere removed at 24 h and processed as described in theconstant exposure method.Free drug retention. For MMAE retention experiments,

25 × 106 cells were seeded at 5 × 105 cells/mL. Each celltype was treated in triplicate with a concentration of radio-active MMAE determined to provide a similar intracellularconcentration for that cell type after 3 h of incubation at37°C. The cells were washed twice into an equal volumeof fresh medium. Each washed culture was split into three15-mL centrifuge tubes. One-milliliter aliquots were re-moved immediately from each tube for LSC and a second1-mL aliquot was layered over 2 mL of FBS in a 15-mLcentrifuge tube, centrifuged at 390 × g for 5 min at roomtemperature. From the separated sample, 0.5 mL of the up-per medium phase was removed for LSC. The remainingportion of each sample was frozen in a dry ice bath andthe bottom of the tube containing the cell pellet was ex-cised into a scintillation vial containing 0.5 mL of PBS.Samples were vortexed; Ecoscint A scintillation fluid(4 mL; National Diagnostics) was added followed by a

Fig. 1. Structure of SGN-35. *, the location of 14C in the radiolabeled drug linker.

Clinical Cancer Research





second vortex; and the samples were counted by LSC(Beckman LS6000IC, Beckman Coulter). Further aliquots(1 mL) were processed over FBS, as described, at the indi-cated time points. For the calculation of intracellular drugconcentrations, cell densities were redetermined by trypanexclusion after washing into nondrug-containing mediumand again after 24 h.Radioactivity calculations. Calculations were made after

subtracting the background from all the disintegration persecond values. The triplicate background-corrected disinte-gration per second readings were averaged and the SD forthose values was calculated using the STDEVPA functionin Microsoft Excel. Average disintegration per second va-lues were converted to μCi and subsequently to pmoleof the drug using the specific activity of the radioactivedrug or drug linker used in the experiment. For each math-ematical manipulation, a propagation of error calculationwas done using standard propagation of error formulas forpropagation of SD values. The intracellular drug concen-trations in nmole were calculated using the estimated vol-ume of 1 × 106 cells.Mass spectrometric drug quantitation. Triplicate samples

of cells were washed into freshmediumat∼5 × 105 cells/mL.ADC was added (200 ng/mL) and the cultures were incu-bated at 37°C in 5% CO2. For intracellular drug quantita-tion, at 24 h, the cells were enumerated by trypan blueexclusion and a known volume of cells was harvested bycentrifugation (360 × g rpm, 5 min, 4°C). The cells werewashed with an equal volume of ice-cold PBS (360 × g,5 min), repelleted, and resuspended in complete culturemedium to provide final volumes of 150 μL. Two volumesof ice-cold methanol were added and the samples werestored at −20°C for ≥30 min. For released drug quantita-tion in the culture medium, cells were prepared in thesame manner; however, aliquots were removed over 3 d.Culture medium was recovered by simple centrifugationof the samples and removal of the supernatant, takingcare not to disturb the pellet. The culture medium samples(150 μL) were mixed with 150 μL of a 50-ng/mL internalstandard in the same culture medium. These were precipi-tated with two volumes of ice-cold acetonitrile. Samples ofmedium and cell pellets not treated with ADCwere used forstandard curves and were prepared following the same pre-cipitation procedures. Eight-point standard curves weremade using varying amounts of MMAE plus constant internalstandard in untreated matrix. All samples were centrifuged athigh speed to remove protein, and the supernatants wereremoved and dried in a centrifugal evaporator.Samples were reconstituted in 33% acetonitrile and

were examined by LC/MS using online solid phase extrac-tion. To derive an equation for the quantitation of releaseddrug in the experimental unknown samples, the peak areafor each drug standard was divided by the peak area ob-tained for the internal standard. The resultant peak arearatios were plotted as a function of the standard concen-trations and the data points were fitted to a curve usinglinear regression. The peak area ratios obtained for the re-leased drug to internal standard in the experimental sam-



ples were converted to drug concentration using thederived equation.ADC-treated culture conditioned medium bioassay. Sam-

ples of spent culture medium from SGN-35–treated cellswere added to CD30-negative Ramos cell cultures in sixdifferent dilutions. In parallel, Ramos cells were incubatedwith eight MMAE concentrations as cytotoxicity standards.After a 96-h incubation at 37°C, the Ramos cultures weredeveloped using resazurin (Sigma; relative fluorescence,excitation = 530-560 nmol/L, emission = 590 nmol/L).Using the average relative fluorescence unit (RFU) mea-surement for Ramos cells incubated with the MMAE stan-dards, the percentage of viable cells relative to untreatedRamos cells (% untreated) was plotted as a function ofMMAE concentration (nmol/L). A four-parameter curvefit was used to generate an equation for the quantitationof released MMAE in the spent culture samples. Cell via-bility measurements of the spent culture dilutions weretransformed into MMAE concentrations (assuming cyto-toxicity is attributed solely to MMAE) using this equation.Only cell viability measurements falling between 15% and85% of the untreated Ramos cells were used to calculatethe concentration of MMAE.Coculture experiments. Karpas299, L540cy, or Ramos

cells in single culture were seeded at 2.5 × 105 cells/mLin culture volumes of 1.5 mL, whereas cocultures ofCD30-positive and CD30-negative pairs consisted of1.25 × 105 cells/mL of each cell type in 1.5 mL of culturemedium (1:1 mixture of cells, RPMI 1640 + 10-15% FBS).The culture medium used in the coculture experiments ad-equately supports the growth of all three cell types. Cul-tures were treated with vehicle control, 1 μg/mL SGN-35,or IgG-vc-MMAE nonbinding control. After a 72-h incuba-tion, cultures were fed with 60% medium containing therequisite treatment type. Cultures were examined for cellcount and viability (Vi-Cell XR2.03 cell viability analyzer,Beckman Coulter) after 120 h and the surviving cells werestained with anti–CD30-Phycoerythrin (BD Biosciences)and anti–CD19-FITC (BD Biosciences) antibodies to deter-mine the distribution of each cell type in the surviving cul-tures. Staining was accomplished by harvesting the cellsby centrifugation at 1,200 rpm for 5 min, plating ∼5 ×105 cells per well in a 96-well plate in 20 μL of fluores-cence-activated cell sorting (FACS) buffer (PBS containing2% FBS), and adding the labeled antibodies to the desiredwells without dilution (5 μL/well). The plate was incubat-ed on ice for 30 min before centrifugation at 1,500 rpm for5 min and before the removal of the supernatant by tap-ping the plate. The cells were washed thrice with PBS(200 μL) before resuspending in 250 μL FACS bufferand before storage at 4°C for subsequent analysis by flowcytometry on a BD FACScan instrument.

Results

Drug release from SGN-35. The kinetics of SGN-35 inter-nalization, drug release, and the extent of intracellular, re-leased drug retention were determined with a radiolabeled




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version of the ADC prepared using 14C-MMAE conjugatedto cAC10 using the mc-valine-citrulline-PABC linker shownin Fig. 1. The resulting ADC contained an average of 4.4MMAEmolecules attached to interchain disulfides as pre-viously described (18, 26). The fate of SGN-35 on culturedcells was examined by the incubation of cells with radiola-beled SGN-35 and the determination of the fraction ofADC in themediumversus associatedwith cells. ADCboundto the cell surfacewas removedbyproteinase K to distinguishit from intracellular ADC. For the media and intracellularfractions, conjugated MMAE was distinguished from re-leased MMAE by precipitation in organic solvent.CD30-positive L540cy HL and Karpas299 anaplastic

large cell lymphoma cells were treated with a constant ex-posure of 14C-labeled SGN-35 at ∼200 times the IC50 con-centration. The total amount of cell-associated drug andintracellular drug are shown in Fig. 2A. Initially, most ofthe drug was associated with the membrane, but intracel-lular bound and released drug built up over the course ofthe assay. Beyond the earliest time point, the vast majority



of intracellular drug was free, suggesting that upon inter-nalization, the release of drug from SGN-35 is quite facile.The concentration of intracellular and extracellular re-

leased drug in cell lines treated with constant SGN-35 ex-posure is shown in Fig. 2B. WSU-NHL cells are CD30negative and did not release detectable levels of drugthrough the entire 3-day course of the assay. In contrast,both CD30-positive cell lines generated released drug,with high intracellular concentrations (>400 nmol/L)reached within 24 hours of treatment. This indicates notonly that SGN-35 was processed in CD30-positive cells,but that the released drug accumulated and was retainedwithin the cells at concentrations much higher than theinitial treatment ADC concentration of 6 nmol/L. Appear-ance of free drug inside and outside the L540cy cells wasgreatly reduced at 4°C or when cells were treated withchloroquine before ADC exposure (Fig. 2C). Chloroquineraises lysosomal pH and reduces the activity of lysosomalproteases having optimal activities under acidic conditions(28). Taken together, these results are consistent with

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Fig. 2. A, generation of MMAE incells treated with MMAE-containingADCs. Radiolabeled MMAEdetected in L540cy and Karpas299cells treated with 200 ng/mL3H-SGN-35. B, intracellular andextracellular concentrations ofsmall-molecule radioactivitydetected in antigen-positive(Karpas299 and L540cy) andantigen-negative (WSU-NHL) cellculture. C, intracellular andextracellular small-moleculeradioactivity detected in L540cyculture after 5 h of 14C-SGN-35treatment (200 ng/mL) at 4°Cand 37°C, with and without100 μmol/L chloroquine (CQ).


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intracellular drug generation by CD30-mediated internali-zation of SGN-35, and accumulation of extracellular drugin the culture medium through escape due to the inherentmembrane permeability of MMAE (29, 30).The combined intracellular and extracellular released

drug over the 72-hour constant exposure incubation pro-vides a basis for estimating the total number of SGN-35molecules that were internalized by the cell and catabo-lized. This number can be compared with the number ofCD30 receptors present in the cell culture at 72 hours andhence provide an estimate of the turnover of CD30 in eachcell line (Table 1). The calculated number of ADCs inter-nalized and catabolized is 2.5 and 3.4 ADCs per receptorfor Karpas299 and L540cy cells, respectively, indicatingthat over the course of the 72-hour SGN-35 incubation,there was either recycling of CD30 to the cell surface orsynthesis of new receptor. This is supported by the amountof ADC that is turned over when cells are treated with alimited exposure to the ADC, in which ice-cold cells areinitially treated with 200 times the IC50 concentration of14C-SGN-35 as in the constant exposure experiment, butsubsequently are washed to remove unbound ADC, pro-viding a treatment condition beginning with only CD30-bound 14C-SGN-35. Under these conditions, approximatelyone ADC is present per receptor. At 24 hours, 0.5 and0.3 ADCs were turned over per receptor in L540cy and Kar-pas299 cells, respectively, compared with 1.2 and 1.1 ADCsper receptor in the same cells under constant exposure con-ditions (Table 1). Synthesis or recycling of the receptorwould be required to obtain an increased number of ADCscatabolized after 24 hours in the constant versus limitedexposure conditions. This finding shows the potential for



receptor recycling or resynthesis in a tumor mass duringthe course of treatment, thus providing new binding sitesfor the remaining SGN-35 present in circulation. Synthesisof new receptor or recycling helps explain the sustainedconcentration of MMAE observed in the CD30-positive cellculture in light of the fact that MMAE is observed to be ac-cumulating in the culture medium (Fig. 2B).Identification of released drug. To determine if the iden-

tity of the released drug was exclusively MMAE, bioassayand mass spectrometry studies were undertaken on thesoluble small molecules generated from CD30-positivecells treated with SGN-35. The data were compared withthe total methanol-soluble radioactivity derived from thecells as shown in Fig. 3A and B. In the bioassay, antigen-negative Ramos cells were treated with the spent culturemedium from SGN-35–treated antigen-positive cells. Us-ing the cytotoxicity of Ramos cells to authentic MMAEstandards, the effective MMAE concentrations in the spentculture samples were calculated. Over time, there was anincreasing antigen-independent cytotoxin concentrationthat overlapped with the presumed MMAE concentrationfrom LSC (Fig. 3A). Furthermore, quantitative mass spec-trometric analysis of released drug in the cell culture super-natants, as well as inside cells (Fig. 3A and B), indicatedthat a species indistinguishable from MMAE by massand retention time was present in concentrations thatalso overlapped with the presumed MMAE concentrationfrom LSC.Cellular efflux of MMAE. Because the escape of released

drug from antigen-positive cells may affect therapeutic effi-cacy, we further established that this type of behavior can beobserved with cells loaded with free MMAE by measuring

Table 1. Catabolism of SGN-35 by CD30-positive cells

L540cy cells (HL)

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Karpas299 cells (ALCL)

CD30*: 587,511/cell
CD30*: 290,676/cell
Limited,†

24 h

Continuous,†

24 h

Continuous,†

72 h

Limited,†

24 h

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Continuous,†

24 h

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n Association for

Continuous,†

72 h

1 × 106 cells/mL

0.53

0.52 ± 0.01

0.33 ± 0.04

0.49

0.65 ± 0.002

0.45 ± 0.02

Intracellular released

MMAE (pmol/mL)

0.31
0.72 ± 0.04 0.62 ± 0.02 0.13 0.49 ± 0.02 0.57 ± 0.03
Extracellular releasedMMAE (pmol/mL)

0.93
2.0 ± 0.1 4.3 ± 0.2 0.24 1.0 ± 0.2 1.9 ± 0.2
Total releasedMMAE (pmol/mL)

1.2
2.7 ± 0.6 4.9 ± 0.2 0.37 1.5 ± 0.1 2.4 ± 0.2
14C-SGN-35 catabolizedper receptor‡

0.54
1.2 ± 0.1 3.44 ± 0.02 0.35 1.1 ± 0.1 2.55 ± 0.01
Abbreviation: ALCL, anaplastic large cell lymphoma.*CD30 levels expressed as the number of mAb molecules bound per cell.†Limited exposure refers to cells treated initially at 0°C with 200 ng/mL 14C-SGN-35 and subsequently washed to remove unboundADC before incubation at 37°C. Continuous exposure refers to treatment of cells at 37°C with 200 ng/mL 14C-SGN-35.‡Calculated using a drug loading of 4.4 drugs/mAb and the average number of cells remaining per milliliter at the time of samplecollection.

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the loss of intracellular drug over 24 hours, during whichtime the cells maintain their viability. To accomplish this,L540cy and Karpas299 cells were treated with sufficient14C-MMAE to achieve intracellular concentrations of ap-proximately 3 to 4 μmol/L and the rate of drug loss wasmeasured, as shown in Fig. 3C. For both cell lines, 50% ofthe initial intracellular MMAE was retained for approxi-mately 16 to 22 hours, and the remaining material wasfound in the culture supernatant. L540cy displays limitedefflux of rhodamine dye whereas Karpas299 does not (Sup-plementary Fig. S1), indicating that the former but not the



latter expresses an unidentified multidrug resistance pro-tein. Despite this difference, the efflux of MMAE from thetwo cell lines is very similar (Fig. 3C), which suggests thatMMAE can diffuse from viable cells regardless of whethera multidrug resistance protein is present. Because MMAEis only observed in the cell culture medium of SGN-35–treated CD30-positive cells and its appearance is inhibitedby the inhibition of lysosomal proteases, the observationthat MMAE can slowly diffuse out of cells bolsters thehypothesis that the extracellular-free drug found in SGN-35–treated culture medium originated from intracellular pro-cessing of the ADC, followed by subsequent passive oractive drug efflux.Bystander effects. Having shown that the released drug

from SGN-35 is MMAE, that the drug is able to diffuseslowly out of the cell from which it was derived(Figs. 2B and 3C), and that SGN-35 is stable under theseculture conditions with no generation of MMAE from an-tigen-negative cell lines (Fig. 2B), we explored whether theADC elicits bystander activity in cocultures with CD30-positive and CD30-negative cell lines. We have previouslyshown (17) that Ramos Burkitt's lymphoma cells areCD30 negative and are as sensitive to free MMAE (IC50,0.04 nmol/L) as Karpas299 (IC50, 0.07 nmol/L) andL540cy (IC50, 0.21 nmol/L) cells, with IC50 values thatare representative of the sensitivity of cancer cells toMMAE. Being CD30 negative, Ramos cells were relativelyinsensitive to SGN-35 (IC50, 3,300 ng/mL with eight drugsper mAb) compared with Karpas299 (IC50, 1.3 ng/mLwith eight drugs per mAb) and L540cy (IC50, 9.9 ng/mLwith eight drugs per mAb) cells (17). FACS analysis of co-cultures of L540cy and Ramos cells showed that treatmentwith 1 μg/mL SGN-35 eliminated both populations ofcells equally well, whereas a similarly treated mixed cellpopulation with a nonbinding control ADC were unaffect-ed (Fig. 4A and B). Similar treatment of the Karpas299/Ramos cell mixture with 1 μg/mL SGN-35 produced thesame outcome (Fig. 4C and D). These results show anantigen-independent cytotoxic effect on the CD30-nega-tive cells that is likely caused by the released MMAE fromthe cocultured CD30-positive cells. A 5-fold lower ADCdose was also examined under the same conditions anddisplayed a less potent reduction of the CD30-negativecells (data not shown), suggesting that the amount ofsmall-molecule cytotoxic agent generated is dependenton the amount of SGN-35 added to the culture. Thus,SGN-35 has strong bystander activity on neighboringantigen-negative cells in culture.

Discussion

Recent advances in ADC research have led to the devel-opment of SGN-35, an agent that has shown considerableactivity, both in preclinical models and in early clinicaltrials (7, 10, 13–16, 18). This molecule incorporates ahighly potent payload, linker technology that is consider-ably more stable than earlier generation hydrazoneand disulfide linkers (7, 8, 31, 32), and conjugation

Fig. 3. Fate of MMAE in cells treated with SGN-35. A, overlay of theextracellular MMAE and small-molecule radioactivity detected by LC/MS,bioassay, and LSC. B, intracellular concentration of MMAE in L540cycells treated with SGN-35 for 24 h detected by LC/MS comparedwith the value of the total small-molecule radioactivity obtained byLSC. C, cellular retention of free MMAE over 24 h.






methodology that provides high yields of well-definedADCs with drugs attached at specific mAb sites that are dis-tal to the variable region (18, 26). The results with SGN-35have provided the basis for extending the findings to in-clude such antigens as LeY (16), CD20 (33), CD22 (30),CD70 (19), CD79b (30), MUC16 (34), EphA2 (35), PSMA(36), and many others. In addition, in vivo model studieshave shown that SGN-35 added to standard chemothera-peutic regimens leads to improved activity over either treat-ment group alone (19). Given the broad applicability ofADCs using this technology, it is of importance to have amolecular understanding of how this ADC functions at amolecular level, the subject of investigations reported here.It has previously been shown that the nature of drug re-

lease from ADCs can have a profound influence on activ-ity. For example, maytansinoid ADCs release lysineadducts if they are linked to mAb lysines throughthioether linkers, but yield S-methylated derivatives iflinked to lysines through disulfide linkers (37). Theseagents have distinct in vitro and in vivo activity profiles,and the charged lysine adduct was shown to be much lessactive than the thioether derivative as a free drug. It wastherefore not surprising that the thioether adduct eliciteda strong bystander effect, whereas the lysine adduct didnot (38). We have also observed that the nature of thelinker can influence the structure of the released drug,based on studies with thioether-linked auristatins that re-leased cysteine adducts through mAb degradation (29).



In the work described here, we show that CD30-positivecell lines process SGN-35 by releasing MMAE in a chemi-cally unmodified form. The results are consistent with theproteolytic cleavage at the citrulline-PABC amide bond,which leads to the release of MMAE after spontaneousfragmentation of the PABC spacer (Fig. 1). This is sup-ported by inhibition studies with chloroquine (Fig. 2C),a lysosomotropic agent that reduces lysosomal proteaseactivity through pH modulation. Upon release, the MMAEdiffuses through cell membranes and accumulates in cul-ture media, albeit at concentrations that were ∼250 timeslower than that found inside the cells, presumably due todilution into the relatively larger volume of medium(Fig. 2B). Despite the great differential, extracellular drugwas able to kill CD30-negative cells cocultured withCD30-positive cells. Thus, SGN-35 has the potential toact on cells within a heterogeneous tumor cell populationthat do not bind sufficiently high amounts of ADC for ef-fective direct cytotoxic activity. In general, this is of impor-tance because mAbs have been shown to distribute withintumors in an uneven manner and many tumors are hetero-geneous with respect to antigen expression (22, 39, 40). Intargeting HL with antibodies directed against CD30 in par-ticular, the ability to eradicate antigen-negative cells with-in the tumor mass may be useful because only a smallpercentage of the cells are CD30 positive and thought tobe a part of the clonal malignancy (25). Because this by-stander cytotoxic effect would be localized to the tumor

Fig. 4. Bystander activities of MMAEcontaining ADCs. Cocultureexperiments included L540cy andKarpas299 (CD30+, CD19−) andRamos (CD30−, CD19+) cells. A, cellcount of L540cy/Ramos coculturestreated with 1 μg/mL SGN-35 and acorresponding nonbinding controlADC (IgG-vc-MMAE). B, L540cy/Ramos cocultures were stained withanti–CD30-PE and anti–CD19-FITCfollowing treatment, allowing thedetermination of the number ofsurviving CD30-positive andCD30-negative cells. C, cell count ofKarpas299/Ramos coculturestreated with 1 μg/mL SGN-35 anda corresponding nonbindingcontrol ADC (IgG-vc-MMAE).D, Karpas299/Ramos cocultureswere stained with anti–CD30-PE andanti–CD19-FITC following treatment,allowing the determination of thenumber of surviving CD30-positiveand CD30-negative cells.




Okeley et al.

896


microenvironment, with cells near the CD30-positive cellsexposed to a concentration gradient of MMAE that de-creases with increasing distance, it is likely to minimize tox-ic systemic exposure of diffusible tumor-releasedMMAE. Insummary, targeted in vitro delivery of MMAE to CD30-expressing cells with SGN-35 leads to high and sustainedintracellular MMAE levels, and successfully ablates bothCD30-expressing malignant cells and neighboring malig-nant cells that do not express the target antigen. This maybe of significance in treating tumors that are heteroge-neous with respect to both antigen presentation andADC distribution (9, 41). The results reported here pro-vide insight into the pronounced activities associated withthis promising ADC.



Disclosure of Potential Conflicts of Interest

All authors are employed by and have ownership interest in SeattleGenetics.

Acknowledgments

We thank Charles G. Cerveny, Damon L. Meyer, and VajiraNanayakkara for the assistance with several of the experimentsdescribed in this article.

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 toindicate this fact.

Received 8/3/09; revised 10/9/09; accepted 12/1/09; publishedOnlineFirst 1/19/10.

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Correction

Correction: Intracellular Activation of SGN-35, aPotent Anti-CD30 Antibody-Drug Conjugate

In this article (Clin Cancer Res 2010;16:888–97), which was published in theFebruary 1, 2010, issue of Clinical Cancer Research (1), there was an error in thelabeling of the legend in the right panel of Fig. 2B. The legend labels for L540cyand Karpas299 in the extracellular MMAE concentration graph were inadvertentlyswitched.

Reference1. Okeley NM, Miyamoto JB, Zhang X, Sanderson RJ, Benjamin DR, Sievers EL, et al. Intracellular

activation of SGN-35, a potent anti-CD30 antibody-drug conjugate. Clin Cancer Res 2010;16:888–97.

Published OnlineFirst August 2, 2011.�2011 American Association for Cancer Research.doi: 10.1158/1078-0432.CCR-11-1753

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Clin Cancer Res; 17(16) August 15, 20115524

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