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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
Note: SuppResearch O
Correspon30th DriveFax: 425-52
doi: 10.115
©2010 Am
Clin Canc
DoDoDo
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].
8/1078-0432.CCR-09-2069
erican Association for Cancer Research.
er Res; 16(3) February 1, 2010
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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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Association for Cancer
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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.
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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
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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
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Released Drug from SGN-35
Published OnlineFirst January 19, 2010; DOI:
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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-
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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
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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
h. 1, 2021. © 2010 American A
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).
Clinical Cancer Research
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Released Drug from SGN-35
Published OnlineFirst January 19, 2010; DOI:
10.1158/1078-0432.CCR-09-2069
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
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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)
h. 1, 2021. ©
Karpas299 cells (ALCL)
CD30*: 587,511/cell
CD30*: 290,676/cell
Limited,†
24 h
Continuous,†
24 h
Continuous,†
72 h
Limited,†
24 h
Clin
2010 America
Continuous,†
24 h
Cancer Res; 16(3) F
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.
ebruary 1, 2010 893
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894
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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
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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.
Clinical Cancer Research
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-
Released Drug from SGN-35
Published OnlineFirst January 19, 2010; DOI:
10.1158/1078-0432.CCR-09-2069
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).
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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.
Clin Cancer Res; 16(3) February 1, 2010 895
h. 1, 2021. © 2010 American Association for Cancer
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Okeley et al.
896
Published OnlineFirst January 19, 2010; DOI:
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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.
Clin Cancer Res; 16(3) February 1, 2010
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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
ClinicalCancer
Research
Clin Cancer Res; 17(16) August 15, 20115524
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2010;16:888-897. Published OnlineFirst January 19, 2010.Clin
Cancer Res Nicole M. Okeley, Jamie B. Miyamoto, Xinqun Zhang, et
al. Antibody-Drug ConjugateIntracellular Activation of SGN-35, a
Potent Anti-CD30
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