apo2l/trail and the death receptor 5 agonist antibody amg 655 cooperate to promote receptor...

Cancer Cell

Article

Apo2L/TRAIL and the Death Receptor 5 AgonistAntibody AMG 655 Cooperate to Promote ReceptorClustering and Antitumor ActivityJonathan D. Graves,1,11 Jennifer J. Kordich,1,12 Tzu-Hsuan Huang,2,14 Julia Piasecki,3 Tammy L. Bush,2 Timothy Sullivan,2

Ian N. Foltz,4 Wesley Chang,5 Heather Douangpanya,1,13 Thu Dang,2 JasonW. O’Neill,6 Rommel Mallari,7 Xiaoning Zhao,7

Daniel G. Branstetter,8 John M. Rossi,9 Alexander M. Long,10 Xin Huang,10 and Pamela M. Holland2,*1Department of Oncology Research, Amgen Inc., Seattle, WA 98119, USA2Therapeutic Innovation Unit, Amgen Inc., Cambridge, MA 02142, USA3Therapeutic Innovation Unit, Amgen Inc., Seattle, WA 98119, USA4Department of Biologic Discovery, Amgen British Columbia, Burnaby, BC V5A 1V7, Canada5Department of Clinical Immunology, Amgen Inc., South San Francisco, CA 94080, USA6Department of Biologic Optimization, Amgen Inc., Seattle, WA 98119, USA7Department of Molecular Structure and Characterization, Amgen, South San Francisco, CA, 94080, USA8Department of Pathology, Amgen Inc., Seattle, WA 98119, USA9Department of Molecular Sciences and Computational Biology, Amgen Inc., Thousand Oaks, CA 91320, USA10Department of Molecular Structure and Characterization, Amgen Inc., Cambridge, MA 02142, USA11Present address: Belmont, MA 02478, USA12Present address: Van Andel Institute, Grand Rapids, MI 49503, USA13Present address: Seattle Genetics, Bothell, WA 98021, USA14Present address: AbbVie, North Chicago, IL 60064, USA

*Correspondence: [email protected]

http://dx.doi.org/10.1016/j.ccr.2014.04.028

SUMMARY

Death receptor agonist therapies have exhibited limited clinical benefit to date. Investigations into whyApo2L/TRAIL and AMG 655 preclinical data were not predictive of clinical response revealed that coadmin-istration of Apo2L/TRAIL with AMG 655 leads to increased antitumor activity in vitro and in vivo. The combi-nation of Apo2L/TRAIL and AMG 655 results in enhanced signaling and can sensitize Apo2L/TRAIL-resistantcells. Structure determination of the Apo2L/TRAIL-DR5-AMG 655 ternary complex illustrates how higherorder clustering of DR5 is achieved when both agents are combined. Enhanced agonism generated bycombining Apo2L/TRAIL and AMG 655 provides insight into the limited efficacy observed in previous clinicaltrials and suggests testable hypotheses to reconsider death receptor agonism as a therapeutic strategy.

INTRODUCTION

Evasion of apoptosis by tumor cells is key to the pathogenesis

and progression of cancer, and the activation of death receptors

has been considered an attractive therapeutic strategy to pro-

mote apoptosis of tumor cells (Ashkenazi et al., 2008; Wiezorek

et al., 2010). Apo2 ligand/tumor necrosis factor (TNF)-related

apoptosis-inducing ligand (Apo2L/TRAIL) is a member of the

Significance

Clinical studies with death receptor agonists have failed to advaered that the DR5 agonist antibody AMG 655 and Apo2L/TRAin vivo, which is associated with enhanced DISC assembly andare sensitized by AMG 655. The structure of the Apo2L/TRAILclustering is achieved in the presence of both agents. These fideath receptor agonists may have demonstrated limited clinpathway activation using Apo2L/TRAIL and AMG 655 in comb

TNF superfamily that induces apoptosis through engagement

of death receptors. Early observations that Apo2L/TRAIL prefer-

entially kills tumor cells over normal cells highlighted its potential

as a candidate therapeutic in cancer and hastened the develop-

ment of monoclonal antibodies selectively targeting death re-

ceptor 4 (DR4) and death receptor 5 (DR5) (Pitti et al., 1996;Wiley

et al., 1995; Ashkenazi et al., 1999). Binding of Apo2L/TRAIL or

agonist antibodies to their cognate death receptors initiates a

nce beyond phase 2 because of limited efficacy. We discov-IL can cooperate to promote tumor cell killing in vitro andapoptotic signaling. Cells resistant to soluble Apo2L/TRAIL-AMG 655-DR5 complex highlights how enhanced receptorndings provide functional and mechanistic insight into whyical activity and provide a strategy to reconsider extrinsicination as a cancer therapeutic.

Cancer Cell 26, 177–189, August 11, 2014 ª2014 Elsevier Inc. 177

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Cancer Cell

TRAIL and AMG 655 Promote Higher Order Clustering

caspase-mediated apoptosis signaling cascade, resulting in cell

death (Pan et al., 1997; Pitti et al., 1996; Sheridan et al., 1997;

Walczak et al., 1997). Although binding of death receptors is pre-

requisite for signal propagation, the level of death receptor

expression in general is a poor predictor of Apo2L/TRAIL sensi-

tivity (Clodi et al., 2000; Kontny et al., 2001; Lincz et al., 2001;

Lippa et al., 2007; Wagner et al., 2007). Not all cancer cells are

sensitive to apoptosis induction via death receptor activation,

and multiple resistance mechanisms have been reported (Yang

et al., 2010). The heterogeneity of resistancemechanisms under-

scores the need to better understand the biological determinants

of responsiveness and how they may be enhanced.

The requirement for receptor aggregation and clustering is a

characteristic shared by many TNF receptor family members

that is essential for efficient signal transduction and may be

one determinant of response (Ashkenazi, 2002). In this regard,

many death receptor agonist antibodies require crosslinking

for optimal activity in vitro and in vivo (Chuntharapai et al.,

2001; Kaplan-Lefko et al., 2010; Wilson et al., 2011). For

example, AMG 655 (conatumumab), a fully human antibody

that selectively targets DR5, has no activity in the absence of

crosslinking, suggesting that receptor oligomerization induced

by crosslinking is required for optimal signal transduction (Ka-

plan-Lefko et al., 2010). Several investigational therapeutics

that target DR4 and/or DR5, including recombinant human

Apo2L/TRAIL (dulanermin), AMG 655, and others, have been

evaluated in clinical studies. However, findings from randomized

phase 2 studies failed to demonstrate significant clinical benefit

(Herbst et al., 2010a; Herbst et al., 2010b; Kindler et al., 2012;

Paz-Ares et al., 2013; Soria et al., 2011; Soria et al., 2010).

Whether these agents underperformed because of insufficient

pathway activation as a result of suboptimal agonism or because

of a lack of integrity of the Apo2L/TRAIL-death receptor pathway

in human tumors remains unclear. In the present study, we

sought to understand why preclinical antitumor activity of AMG

655 and Apo2L/TRAIL was not predictive of clinical efficacy.

RESULTS

Apo2L/TRAIL and AMG 655 Cooperate In Vitro andIn VivoTo elucidate the relationship between Apo2L/TRAIL and AMG

655 for binding and agonism of DR5, we used a cell-based

viability assay using WM35 cells. WM35 cells express surface

DR5 but undetectable levels of DR4, ensuring DR5-specific

signaling with Apo2L/TRAIL (Figures S1A and S1B available

online). Because AMG 655 binds to DR5 but does not signal

without additional crosslinking (Kaplan-Lefko et al., 2010), pre-

treatment of WM35 cells with AMG 655 followed by treatment

with Apo2L/TRAIL would allow the functional relationship be-

tween the two agents for DR5 binding and agonism to be deter-

mined. Although AMG 655 in the absence of protein G had no

effect, either AMG 655 plus protein G (which provides maximal

crosslinking) or Apo2L/TRAIL induced dose-dependent loss of

viability in WM35 cells (Figure 1A). Unexpectedly, pretreatment

with AMG 655 (in the absence of protein G) markedly enhanced

killing induced by Apo2L/TRAIL. This effect was more potent

than leucine zipper (LZ)-Apo2L/TRAIL, a nonoptimized form of

the ligand known to be a potent agonist (Lawrence et al., 2001;

178 Cancer Cell 26, 177–189, August 11, 2014 ª2014 Elsevier Inc.

Walczak et al., 1999). Because the order of addition did not

significantly influence their cooperative relationship (data not

shown), Apo2L/TRAIL and AMG 655 were added simultaneously

in subsequent experiments.

We next tested a panel of monoclonal antibodies to determine

whether DR5 agonist antibodies other than AMG 655 could

cooperate with Apo2L/TRAIL. Treatment of WM35 cells with

M413, previously shown to compete with LZ-Apo2L/TRAIL for

binding to DR5 (Griffith et al., 1999), or M411 blocked Apo2L/

TRAIL-mediated killing (Figure 1B). Despite a demonstrated abil-

ity to bind DR5, other antibodies either had no effect or were able

to cooperate with Apo2L/TRAIL, suggesting that only a subset of

anti-DR5 antibodies can cooperate with Apo2L/TRAIL.

To determine the prevalence of cooperativity between Apo2L/

TRAIL and AMG 655, we evaluated panels of breast, colon, and

lung cancer cell lines. We found that a high proportion of cells

that were sensitive to either single agent demonstrated

enhanced sensitivity to the combination (Figure 1C; Figure S1C).

All cells expressed both DR4 and DR5, and there was no strong

correlation with sensitivity and receptor expression levels (Fig-

ure S1B). We also tested whether Apo2L/TRAIL and AMG 655

would cooperate in vivo. In mice bearing SW403 xenograft

tumors, only the combination of Apo2L/TRAIL and AMG 655

resulted in significant antitumor activity (Figure 1D), demon-

strating that Apo2L/TRAIL and AMG 655 show synergistic

activity in vivo as well as in vitro. In addition, we tested Apo2L/

TRAIL and AMG 655 cooperativity in an H460 lung adenocarci-

noma xenograft model, which has been shown to be partially

sensitive to Apo2L/TRAIL (Jin et al., 2004). To confirm that the

in vivo cooperativity of AMG 655 and Apo2L/TRAIL was inde-

pendent of Fcg receptor (FcgR) interactions, we included an

aglycosylated variant of AMG 655 (N297A-AMG 655) that is

unable to bind FcgRs and therefore lacks intrinsic activity. Muta-

tion of the invariant glycan at N297 in the antibody Fc domain

is known to dramatically reduce the binding affinity to Fc recep-

tors (Jung et al., 2011). When we compared the efficacy of AMG

655 or N297A-AMG 655 in the presence or absence of Apo2L/

TRAIL, we observed partial tumor growth inhibition with

Apo2L/TRAIL and no antitumor activity with either AMG 655 or

the N297A -AMG 655 variant (Figure S1D). However, tumor

growth inhibition in both combination treatment groups was

significantly greater than Apo2L/TRAIL, indicating that both

AMG 655 and N297A-AMG 655 can cooperate with Apo2L/

TRAIL in vivo and cooperativity is independent of FcgR-medi-

ated crosslinking.

During the profiling of cell lines in vitro, we noted several exam-

ples of strong cooperativity in cells that were resistant to Apo2L/

TRAIL but responsive to AMG655when crosslinked by protein G

(Figure 2A). The ability of AMG 655 (plus protein G) to induce

apoptosis suggests that the death receptor signaling pathway

is intact but that soluble Apo2L/TRAIL is not an optimal agonist

in this subgroup of cells. To test this hypothesis we cocultured

293T cells or 293T cells expressing the membrane form of

Apo2L/TRAIL (Figure 2B) with cell lines that were resistant to

soluble Apo2L/TRAIL but sensitive to Apo2L/TRAIL plus AMG

655. Although there was no effect in response to soluble

Apo2L/TRAIL (Figure 2C), we observed a dose-dependent in-

crease in caspase-3/7 activity in the presence of 293T-TRAIL

cells (Figure 2D; Figure S2). These findings indicate that

Figure 1. Apo2L/TRAIL and AMG 655 Cooperate in Multiple Cell Lines In Vitro and In Vivo

(A) WM35 cells were treated with indicated agents for 24 hr and assayed for cell viability. For combinations, protein G and AMG 655 were at 1 mg/ml.

(B) WM35 cells were treated with Apo2L/TRAIL and 1 mg/ml of the indicated antibodies for 24 hr and assayed for cell viability.

(C) Viability of breast (left) or lung (right) tumor lines 24 hr after treatment with Apo2L/TRAIL + hIgG1 (1 mg/ml), Apo2L/TRAIL, AMG 655 plus protein G (1 mg/ml), or

Apo2L/TRAIL plus AMG 655 (1 mg/ml).

(D) Mice bearing SW403 tumors were treated with Apo2L/TRAIL (60 mg/kg, five times per week, i.p.), AMG 655 (100 mg/mouse, twice per week, i.p.), or the

combination for two cycles (n = 10/group). *For Apo2L/TRAIL plus AMG 655, p < 0.0001 versus PBS by Dunnett’s test.

Data are presented as mean ± SEM for all panels. See also Figure S1.

Cancer Cell


membrane-associated Apo2L/TRAIL was more effective at

inducing DR5 crosslinking and clustering than the soluble form

and suggest that the coadministration of Apo2L/TRAIL with

AMG 655 may functionally mimic a membrane-bound form of

Apo2L/TRAIL, providing not only a quantitative but also a quali-

tative change in how the signal is perceived.

Apo2L/TRAIL and AMG 655 Promote ReceptorOligomerization and Higher Order ClusteringTo address the mechanistic basis of cooperativity between

Apo2L/TRAIL and AMG 655, we first tested whether AMG 655

must be bivalent in order to synergize with Apo2L/TRAIL.

Although AMG 655 or a Fab02 fragment could enhance

Apo2L/TRAIL-mediated apoptosis, a Fab fragment of AMG

655 could not (Figure 3A). These data support the hypothesis

that the mechanism of cooperativity between AMG 655 and

Apo2L/TRAIL likely involves enhanced receptor crosslinking

and oligomerization. We then asked whether Apo2L/TRAIL

and AMG 655 could be coimmunoprecipitated. After treatment

of Colo205 cells with Apo2L/TRAIL, AMG 655, or the combina-

tion, lysates were immunoprecipitated with protein G (to pull

down AMG 655) and immunoblotted for the presence of

Apo2L/TRAIL and DR5. Significantly more DR5 coimmunopre-

cipitated with AMG 655 in the presence of Apo2L/TRAIL, sug-

gesting the formation of a ternary complex on Colo205 cells

(Figure 3B; Figures S3A and S3B). We also evaluated whether

the coadministration of Apo2L/TRAIL and AMG 655 resulted

in enhanced formation of a death-inducing signaling complex

(DISC), a critical step in signal initiation by oligomerized death

receptors. AMG 655 augmented the formation of a complex


Figure 2. Cells Resistant to Soluble Apo2L/TRAIL Are Sensitive to Membrane-Bound Apo2L/TRAIL

(A) SkLu-1, SW403, and SW1573 cells were treated with the indicated agents for 24 hr and assayed for cell viability. For combinations, hIgG1, protein G, and AMG

655 were at 1 mg/ml. Data are presented as ±SEM.

(B) Staining of 293T cells or 293T cells transfected with control vector or a vector expressing Apo2L/TRAIL (293T-TRAIL) was performed with anti-TRAIL antibody

(M180), and cells were analyzed using flow cytometry.

(C) SkLu-1, SW403, and SW1573 cells (23 104 cells/well) were treated with soluble Apo2L/TRAIL as indicated for 4 hr and assayed for caspase-3/7 activity. Data

are presented as ±SEM.

(D) Coculturing of 293T or 293T-TRAIL cells was performed with SkLu-1, SW403, or SW1573 cells at the indicated ratio for 4 hr, and cells were assayed for

caspase-3/7 activity.

Data are shown as mean ± SEM and are representative of at least three independent experiments. See also Figure S2.

Cancer Cell


between FADD, caspase-8, and DR5 upon stimulation of either

Colo205 or SkLu-1 cells with Apo2L/TRAIL (Figure 3C; Fig-

ure S3C). Further support for this hypothesis was provided by

confocal microscopy, showing that only coadministration of

Apo2L/TRAIL and AMG 655 resulted in the formation of large

clusters containing AMG 655 and cleaved caspase-8 (Fig-

ure 3D), reinforcing the conclusion that these enhanced clusters

represent functional signaling complexes. Taken together,

these data indicate that the cooperative induction of apoptosis

by Apo2L/TRAIL and AMG 655 is due to increased DISC forma-

tion as a consequence of enhanced DR5 crosslinking. To

ensure that Apo2L/TRAIL and AMG 655 were incorporated

into a ternary complex on cells, we performed a gel filtration

analysis. WM35 cells were treated with AMG 655, Apo2L/

TRAIL, or the combination, and lysates were separated by

size-exclusion chromatography and immunoblotted for the

presence of DR5. Figure 3E shows that DR5 elutes in the higher

molecular weight fractions only when Apo2L/TRAIL and AMG

655 are coadministered, indicating the formation of a higher

order complex.


To better understand the molecular interactions between

Apo2L/TRAIL, DR5, and AMG 655, we determined the crystal

structure of the ternary complex of Apo2L/TRAIL, the DR5 ecto-

domain, and the Fab fragment of AMG 655 at 3.3 A resolution

(Table 1). The Apo2L/TRAIL-DR5-AMG 655-Fab complex is

composed of an Apo2L/TRAIL homotrimer with three DR5

monomers bound diagonally along the crevices between two

neighboring Apo2L/TRAIL molecules and three AMG 655 Fabs

bound to the three DR5 monomers individually (Figure 4A).

Although there is extensive interaction between AMG 655 and

DR5, we did not observe any atomic contacts between AMG

655 and Apo2L/TRAIL. The AMG 655-DR5 interface covers an

area of approximately 1,249 A2, split evenly between AMG 655

(644 A2) and DR5 (605 A2). Residues of all six complemen-

tarity-determining regions (CDRs) of AMG 655 are involved in

the interaction with DR5 (Figure 4B). The AMG 655 binding site

on DR5 consists of 11 residues, forming a nearly contiguous

region in the cysteine-rich domain 2 (Figure 4C; Figure S4). The

structure of DR5 and Apo2L/TRAIL in the ternary complex super-

imposes well with the structure of the binary Apo2L/TRAIL-DR5

Figure 3. Apo2L/TRAIL and AMG 655 Promote

Receptor Oligomerization and Increased Signaling

(A) Colo 205 cells were treated with the indicated agents

and analyzed for cell viability after 6 hr. Fixed molar

amounts of AMG 655, AMG 655 Fab, or AMG 655 Fab02were added. Data are presented as ± SEM.

(B) Lysates from Colo205 cells treated for 30 min with

media (C), 1 mg/ml AMG 655 (655), 1 mg/ml Apo2L/TRAIL

(Apo), 1 mg/ml Apo2L/TRAIL plus 1 mg/ml human IgG (Apo

IgG), or 1 mg/ml Apo2L/TRAIL plus 1 mg/ml AMG 655 (Apo

655) were immunoprecipitated using protein G (to pull

down AMG 655) and immunoblotted using anti-hIgG (to

detect AMG 655), anti-DR5, or anti-TRAIL antibody.

(C) Lysates from Colo205 (left) or SkLu-1 (right)

cells treated for 4 hr with Apo2L/TRAIL (Apo) (Colo205

1.6 ng/ml, SkLu-1 1 mg/ml), 1 mg/ml AMG 655 (655), or the

combination (Cb) were immunoprecipitated using mouse

anti-FADD antibody (or IgG, far right lane) and immuno-

blotted using anti-DR5, anti-caspase-8 (C8), or anti-FADD

antibody.

(D) SkLu-1 cells treated with 1 mg/ml Apo2L/TRAIL (left),

1 mg/ml AMG 655 (center), or the combination (right) for

15 min were stained with DAPI (blue) or antibodies against

human IgG (red) to visualize AMG 655 or cleaved C8

(green). White triangles indicate the colocalization of AMG

655 and cleaved C8. The scale bar represents 4 mm.

(E) WM35 cells were treated for 15 min with 1 mg/ml AMG

655, 1 mg/ml Apo2L/TRAIL, 1 mg/ml Apo2L/TRAIL plus

1 mg/ml human IgG, or 1 mg/ml Apo2L/TRAIL plus 1 mg/ml

AMG 655. Total soluble protein from cell lysates was

separated by size-exclusion chromatography, and frac-

tions 2 to 11 from each treatment were immunoblotted for

the presence of DR5. (Top) UV absorption profile, which

was identical for all lysates. Numbers above the peaks

correspond to molecular weights from a standard.

Numbers 1 to 12 correspond to elution of indicated

fractions. Elution volume is noted on the x axis. mAU =

milli-absorbance units. For western blots, the first lane

corresponds to WM35 total lysate before fractionation.

See also Figure S3.

Cancer Cell



Table 1. Apo2L/TRAIL-DR5-AMG 655 Structural Data Collection

and Refinement Statistics

Apo2L/TRAIL-DR5-AMG 655 Fab

Data Collection

Space group P6122

Cell dimensions

a, b, c (A) 152.01, 152.01, 613.16

a, b, g (�) 90, 90, 120

Resolution (A) 3.30

Rsym or Rmerge 0.166 (0.698)

I/sI 9.4 (2.25)

Completeness (%) 98.9 (99.9)

Redundancy 4.3 (4.4)

Refinement

Resolution (A) 3.30

No. of reflections 60,562

Rwork/Rfree 0.229/0.285

No. of atoms

Protein 15,933

Ligand/ion 1

Water

B-factors

Protein 75.39

Ligand/ion 57.08

Water

Rmsds

Bond lengths (A) 0.0099

Bond angles (�) 1.61

One crystal was used to obtain the structure. Values in parentheses are

for highest-resolution shell.

Cancer Cell


complex (Hymowitz et al., 1999; Mongkolsapaya et al., 1999),

suggesting that binding of AMG 655 to DR5 does not alter the

conformation of either DR5 or Apo2L/TRAIL.

To infer the structure of full-length AMG 655, we superim-

posed the three Fab fragments in the ternary complex with Fab

fragments from previously determined full-length immunoglob-

ulin G1 (IgG1) antibody structures (Guddat et al., 1993; Saphire

et al., 2001). On the basis of the AMG 655 Fab conformation,

we predict that the Fc domain is pointing down toward the cell

surface (Figure 4D). Despite this orientation, our functional

studies support that the Fc domain of AMG 655 is available for

engagement with protein G and Fc receptors, and this may be

afforded by the inherent flexibility within the antibody hinge

region. We then modeled the positioning of three additional

ternary complexes, which are situated in the same plane as the

original ternary complex, parallel to the cell membrane (Fig-

ure 4E). Further addition of ternary complexes results in an array

of DR5 molecules on the cell surface in a hexagonal honeycomb

pattern, with three DR5 molecules in each node (Figure 4F). This

model illustrates how co-administration of trimeric Apo2L/TRAIL

and bivalent AMG 655 may promote superclustering of DR5,

thereby leading to efficient DISC assembly and enhanced

apoptotic signaling.


Apo2L/TRAIL and aMouseDR5Antibody AreEfficaciousat Tolerated DosesMice harbor only one death receptor for Apo2L/TRAIL, mouse

DR5 (mDR5)/muTRAILR2 (Takeda et al., 2007). Whereas AMG

655 does not bind to mDR5, MD5-1, a monoclonal antibody

that selectively recognizes mDR5, exhibits killing activity on

mouse tumor cells upon crosslinking in vitro (Takeda et al.,

2004). We reasoned that MD5-1 may have similar crosslinking

requirements to AMG 655 and therefore may also cooperate

with Apo2L/TRAIL. We treated murine tumor cell lines with

Apo2L/TRAIL and MD5-1 in vitro and found that the majority of

lines were efficiently killed only when Apo2L/TRAIL and MD5-1

were combined (Figure 5A). All of the lines expressed similar

levels of mDR5 (Figure S5A). We selected two cell lines, Renca

and LLC1, to test for Apo2L/TRAIL and MD5-1 cooperativity

in vivo. In both models, we observed marked antitumor activity

in the presence of Apo2L/TRAIL and MD5-1 but not with either

agent alone (Figure 5B; Figures S5B–S5D). Tumors were har-

vested for histological analysis 3 days after the start of treatment.

Upon macroscopic observation, tumors treated with Apo2L/

TRAIL and MD5-1 displayed a hemorrhagic appearance (Fig-

ure 5C). Histological evaluation as early as 8 hr after treatment

revealed that in contrast to either agent alone, tumor cells as

well as tumor-associated vasculature from mice treated with

Apo2L/TRAIL and MD5-1 were positive for activated caspase-

3, suggesting that both tumor cells and the surrounding vascula-

ture underwent apoptosis after treatment with Apo2L/TRAIL and

MD5-1 (Figure 5D; Figure S5E). Indeed, we observed strong

expression of mDR5 on both tumor cells and associated vascu-

lature but, interestingly, could not detect mDR5 expression on

vasculature in other tissues (Figure 5E; Figure S5F). Disruption

of tumor-associated vasculature by crosslinked Flag-Apo2L/

TRAIL, but not soluble Apo2L/TRAIL, was previously reported

(Wilson et al., 2012). Our results are consistent with previous

findings in that soluble Apo2L/TRAIL alone had no effect, but

oligomeric forms of Apo2L/TRAIL, either by Flag-mediated

crosslinking (Wilson et al., 2012) or by coadministration with

MD5-1, can induce antitumor activity by impacting tumor cells

directly, as well as tumor-associated vasculature where mDR5

is expressed.

We did not observe any evidence of toxicity in tissues exam-

ined from syngeneic tumor efficacy studies (Figure S5G). How-

ever, coadministration of Apo2L/TRAIL andMD5-1 but not either

agent alone to naive mice at high doses resulted in decreased

body weight, associated with diarrhea, dehydration, and scruffy

coat over the course of one cycle. Upon necropsy, gross obser-

vations were noted in the stomach and gastrointestinal (GI) tract

in the combination treatment group only. Histological analysis

indicatedminimal to severe apoptosis, degeneration, and necro-

sis in the epithelium of tissues that expressed the highest levels

of mDR5 only when Apo2L/TRAIL and MD5-1 were coadminis-

tered. This effect was observed predominantly in the stomach,

duodenum, cecum, colon, and gallbladder (Figures S5H and

S5I). There was evidence of minimal hepatocyte vacuolation in

the liver, but no mDR5 expression was detectable in liver by

immunohistochemistry (Figure S5I). Our findings from efficacy

studies in syngeneic models and high-dose tolerability studies

indicate that a therapeutic window can be achieved when

Apo2L/TRAIL and MD5-1 are coadministered in mice, and

Figure 4. Crystallographic Analysis of the

Apo2L/TRAIL-DR5-AMG 655 Fab Ternary

Complex

(A) Ribbon representation of the Apo2L/TRAIL-

DR5-AMG 655 Fab complex with view down the

3-fold axis. Apo2L/TRAIL (blue) with Zn atom (red)

binds three DR5 monomers (tan, space-filling

mode), and each DR5 monomer binds one AMG

655 Fab fragment (light chain, yellow; heavy chain,

green) individually.

(B) Locations of DR5 and AMG 655 residues

involved in binding (red). CDRH1-H3 correspond to

heavy chain, and CDRL1-L3 correspond to light

chain.

(C) The AMG 655 binding site (orange) on DR5 (tan)

involves residues K98, Y99, Q101, R118, C119,

S121, E123, V124, E125, P128, and E140. The

Apo2L/TRAIL binding region is shown in purple.

(D) Inferred structure of full-length AMG 655 in

complex with Apo2L/TRAIL and DR5 by super-

position with previously determined structures of

IgG1 antibodies. The Fc domain of AMG 655

(orange) is predicted to point down toward the cell

surface.

(E) View of three Apo2L/TRAIL-AMG 655-DR5

ternary complexes down the 3-fold axis. The AMG

655 Fc domain points down into the page.

(F) Model for induction of higher order clustering by

multiple ternary complexes. The image in (A) is

represented in the black box. Red dots correspond

to Apo2L/TRAIL bound to three DR5 receptors.

Blue lines denote AMG 655 Fab fragments ex-

tending from DR5.

See also Figure S4.

Cancer Cell


potential target tissues for toxicity are associated with higher

mDR5 expression. To understand whether DR5 expression

was conserved across species, we evaluated a subset of tissues

from mouse, human, and nonhuman primate, with emphasis on

those that exhibited high mDR5 expression in mouse. As in the

mouse, DR5 expression was more abundant in the GI tract

tissues of human and nonhuman primate compared with other

tissues examined (Figure S5J).

Apo2L/TRAIL and AMG 655 Are Efficacious againstPrimary Human Tumor CellsTo determine whether our findings might have clinical relevance,

we tested Apo2L/TRAIL plus AMG 655 on fresh primary tumor

disaggregates from 12 lung carcinoma patients (Table S1). We

initially evaluated the degree of Apo2L/TRAIL and receptor

expression on tumor infiltrating CD45+ cells and EpCAM+ tumor

cells. CD45+ cells expressed higher amounts of the decoy recep-

tors DcR1 and DcR2, whereas EpCAM+ cells expressed mostly

DR5 and DcR1 (Figure 6A). Tumor disaggregates were then

treated with AMG 655 or Apo2L/TRAIL alone or in combination,

and caspase-3 activity was measured after 18 hr (Figure 6B).

Cancer Cell 26, 177–18

There were varying degrees of sensitivity

among the lung tumor samples tested

and a trend toward increased responsive-

ness in the presence of both agents

versus either agent alone.

We also tested whether Apo2L/TRAIL plus AMG 655 might

have killing activity on nonmalignant primary cells. All of the

nonmalignant cells tested expressed DR5, and although some

activity was noted in hepatocytes and keratinocytes from

different donors, it was not consistent and did not approach

the degree of cooperativity observed on tumor cell lines (Fig-

ure 6C; Figure S6). No combinatorial effect was observed on

monocytes, renal epithelial cells, endothelial cells, or fibroblasts

from multiple donors.

DISCUSSION

The therapeutic potential of death receptor agonist antibodies

or soluble Apo2L/TRAIL has not yet been realized in a clinical

setting. For agonist antibodies, one possible reason may be

the crosslinking requirement for effective receptor engagement

and clustering. In vivo, crosslinking is mediated by FcgRs

expressed on the surface of immune cells. Both drozitumab

and AMG 655 are dependent on such crosslinking for activity

(Kaplan-Lefko et al., 2010; Wilson et al., 2011). Moreover,

in phase 2 trials conducted with AMG 655, a trend toward a

9, August 11, 2014 ª2014 Elsevier Inc. 183

Figure 5. Murine Tumor Cells Are Sensitive to Apo2L/TRAIL and MD5-1 In Vitro and In Vivo

(A) Murine tumor lines treated with Apo2L/TRAIL, Apo2L/TRAIL plus hamster IgG (1 mg/ml), MD5-1 or Apo2L/TRAIL + MD5-1 (1 mg/ml) were assayed for cell

viability after 48 hr. Data are shown as mean ± SEM and are representative of at least three independent experiments.

(B) Renca tumor-bearing mice were treated with Apo2L/TRAIL (5 mg/kg, five times per week), MD5-1 (1 mg/mouse, once per week), or the combination for two

cycles (n = 10/group). *For Apo2L/TRAIL plus MD5-1, p < 0.0001 versus PBS by Dunnett’s test. Three of ten tumors in the combination group had nomeasurable

tumor. Data are presented as mean ± SEM.

(C) Representative tumors from macroscopic analysis of PBS or Apo2L/TRAIL plus MD5-1 treated tumors 3 days after the start of treatment.

(D) Representative tissues from Renca tumor-bearing mice treated with PBS or Apo2L/TRAIL plus MD5-1, 8 hr after treatment, stained with antibody for active

caspase-3.

(E) Representative tissues stained with antibody for mDR5. For both (D) and (E), Apo2L/TRAIL and MD5-1 single-agent groups were indistinguishable from PBS-

treated mice (data not shown). Individual vessels are denoted by black arrows. The scale bars represent 100 mm (upper left panel) and 50 mm (other panels).

See also Figure S5.

Cancer Cell


stronger treatment effect was observed in patients carrying

a high-affinity allele of the FcgRIIIa receptor, suggesting

that the high-affinity form of the receptor may be associated

with enhanced responsiveness (Pan et al., 2011). Therefore,

insufficient crosslinking could contribute to a diminished

antitumor response mediated by death receptor agonist

antibodies.


The limited clinical efficacy observed with Apo2L/TRAIL could

be attributed to its short half-life (Herbst et al., 2010a), but addi-

tional limitationsmay be associatedwith its soluble form. Several

soluble TNF family ligands show different activity profiles from

their corresponding membrane-associated forms. For example,

FASL has weak apoptosis-inducing activity as a soluble form

(O’Reilly et al., 2009; Schneider et al., 1998; Suda et al., 1997)

Figure 6. Activity of Apo2L/TRAIL and AMG 655 on Primary Tumor and Nonmalignant Cells

(A) Freshly resected lung tumor-derived cell suspensions were stained with antibody cocktails against Apo2L/TRAIL, DR4, DR5, DcR1, or DcR2 with either

EpCAM (top) or CD45 (bottom). The symbol on each graph represents an individual tumor sample. Black horizontal lines represent mean expression.

(B) A gating strategy to identify EpCAM+ events was used, and samples were treated with the indicated agents (IgG1, 1 mg/ml; AMG 655, 1 mg/ml; Apo2L, 2 mg/ml;

Apo2L lo, 0.13 ng/ml; Apo 2L med, 1.6 ng/ml; Apo 2L hi, 2 mg/ml) for 18 hr and analyzed for caspase-3/7 activity. Results are shown as percentage viable events

remaining in treated samples relative to vehicle. Black horizontal lines represent mean expression. For one sample (black squares), there was not enoughmaterial

to run Apo2L plus IgG1 control.

(C) Viability of primary human cells treated with Apo2L/TRAIL, AMG 655 plus protein G (1 mg/ml), or the combination (AMG 655, 1 mg/ml) for 48 hr (monocyte,

human umbilical vein endothelial cell, fibroblast, epithelial, keratinocyte) or 72 hr (hepatocyte).

Data are presented as mean ± SEM. See also Figure S6 and Table S1.

Cancer Cell


but can cooperate with an agonistic anti-FAS receptor antibody

to kill target cells (Xiao et al., 2002). Others have also described

limited responses with soluble Apo2L/TRAIL compared with

membrane-associated or oligomerized forms (Berg et al.,

2007; Muhlenbeck et al., 2000; Wajant et al., 2001). This has

prompted the development of alternative death receptor ago-

nists engineered to promote enhanced receptor clustering.

Many approaches to either improve the stability of Apo2L/TRAIL

or overcome the limitations of bivalent antibodies have been

reported (de Bruyn et al., 2013; Gieffers et al., 2013; Huet

et al., 2012; Pavet et al., 2010; Pfizenmaier and Szymkowski,

2011; Swers et al., 2013).

Coadministration of Apo2L/TRAIL and AMG 655 potently kills

tumor cells without a requirement for additional crosslinking. We

propose that the additional receptor crosslinking provided by

AMG 655 changes the signal delivered by soluble Apo2L/TRAIL

into one that functionally resembles membrane-associated

Apo2L/TRAIL. The failure of soluble Apo2L/TRAIL to appropri-

ately engage receptors may have implications into its limited

clinical activity. Similarly, the efficacy of AMG 655 may have


Cancer Cell


been limited by insufficient crosslinking by Fc receptor-bearing

cells in the tumor. Importantly, AMG 655 lacks activity in the

absence of an exogenous crosslinker such as protein G, and

protein G-mediated cross-linking in vitro is likely an overrepre-

sentation of the extent of crosslinking that occurs in vivo. Thus,

the dose response of single-agent AMG 655 (plus protein G) is

not reflective of the contribution of AMG 655 in the context of

soluble Apo2L/TRAIL, and, importantly, agonism by the combi-

nation is independent of the need for crosslinking (i.e., by Fc re-

ceptors in vivo).

Despite the enhanced killing activity of Apo2L/TRAIL and AMG

655 administered together, not all cell lines could be sensitized

by the combination. Current understanding of mechanisms of

resistance suggests thatmultiple factors contribute to a resistant

phenotype. These include receptor expression, the ability of

the receptor to initiate a signal, and the threshold sensitivity of

the cell to the initiating signal, which may be dependent on the

expression of other pro- and antiapoptotic factors. We have

observed that cells that are resistant to combination Apo2L/

TRAIL plus AMG 655 are also resistant to soluble Apo2L/TRAIL

and AMG 655 (plus protein G) individually. We believe this pop-

ulation likely includes cells that lack pathway integrity and for

which agonism is not a limiting factor. In contrast, cells that

have an intact pathway butmay be below the threshold for sensi-

tivity are potently sensitized by the combination.

Wepresent the structure of aTNF receptor in a ternary complex

with its ligand and an agonist antibody. The X-ray structure of the

Apo2L/TRAIL-DR5-AMG 655 Fab ternary complex provides a

unique opportunity for studying the molecular mechanism of

DR5 activation by Apo2L/TRAIL and AMG 655. Although only

the Fab fragment of AMG 655 was used for crystallization, we

could infer the structure of AMG 655 IgG from previously deter-

mined human IgG1 antibody structures. Our structural data pro-

vide the basis for amodel depicting how a ternary complex forms

andhow recruitment of additional ternary complexes can result in

enhanced DR5 clustering and signaling. This is consistent with

the model of DISC formation through clustering represented by

the FAS-FADD death domain complex structure (Scott et al.,

2009). Similar findings based on structure have been described

for the facilitation of TRAF6-mediated ubiquitination, where

TRAF6 dimerization induces higher order oligomerization (Yin

et al., 2009). Both this model and ours describe a proposed scaf-

fold for higher order oligomerization thatmay facilitate ubiquitina-

tion or proximity-induced caspase activation. It was recently

proposed that Apo2L/TRAIL-induced cell death is triggered via

ligand-induced DR5 dimerization to generate organized ligand-

receptor networks (Valley et al., 2012). In our model, the addition

of AMG 655 serves to stabilize Apo2L/TRAIL-DR5 complexes by

bridging between receptors, thereby promoting enhanced clus-

tering and signaling.

Our structural analysis also provides insight into the improved

killing activity observed both in vitro and in vivo. Importantly,

coadministration of Apo2L/TRAIL with the murine mDR5 anti-

body MD5-1 resulted in strong antitumor activity. The coopera-

tivity of Apo2L/TRAIL and MD5-1 provided an opportunity to

test the effects of enhanced signaling in a model in which safety

margins could be assessed. At high doses (200 times higher

MD5-1 and 12 times higher Apo2L/TRAIL than used in the effi-

cacy studies), we saw evidence of toxicity in areas where


mDR5 was most abundantly expressed, notably in tissues of

the GI tract. Analysis of DR5 expression in other species also

showed the highest expression in GI tract tissues. Collectively,

this supports the idea that despite the increased potency of

Apo2L/TRAIL and AMG 655, a therapeutic window may still

be achievable, and evaluation of combination agonists in a

nonhuman primate may be predictive of tolerability. As previ-

ously reported for an oligomerized form of Apo2L/TRAIL, we

also noted effects specifically on tumor vasculature, where

mDR5 was abundantly expressed. It is not clear whether

Apo2L/TRAIL and AMG 655 would exert similar effects, as

AMG 655 and MD5-1 may have unique properties. Moreover, lit-

tle is known about DR5 expression in human tumor-associated

vasculature. In previous reports, endothelial DR5 expression in

human lung tumor specimens was found to be rare (Wilson

et al., 2012).

Notably, we observed a trend toward increased sensitivity

to Apo2L/TRAIL and AMG 655 in primary human lung tumor

samples compared with either agent alone. Treatment with

AMG655 alone or Apo2L/TRAIL up to 2 mg/ml resulted inminimal

activity. In contrast, even small amounts of Apo2L/TRAIL

(<2 ng/ml) were sufficient to cooperate with AMG655 in sensitive

samples. Although there was variable sensitivity to the combina-

tion across the lung samples profiled, these findings highlight the

potential for improving the likelihood of response in a patient

setting by coadministration of Apo2L/TRAIL and AMG 655.

In summary, the quantitatively and qualitatively enhanced

agonism generated by combining Apo2L/TRAIL and AMG 655

provides insight into the limited activity in previous clinical trials.

We propose that soluble Apo2L/TRAIL and AMG 655 are subop-

timal agonists as single agents because of an inability to induce

sufficient receptor clustering and activation. In our model, solu-

ble Apo2L/TRAIL can efficiently induce the formation of active

receptor trimers but is less efficient at promoting their clustering,

thereby producing insufficient signaling. The bivalent nature of

AMG 655 means that it is unlikely to be an efficient inducer of re-

ceptor trimers. However, its contribution in the combination

setting may be to bridge receptors to promote clustering and

productive signaling. This model provides possible explanations

for the limited activity of both Apo2L/TRAIL and AMG 655 that

are unified by a common aspect of TNF receptor biology: the

need for receptor clustering. Furthermore, in the context of a

combination that is both potent and independent of exogenous

crosslinking, Apo2L/TRAIL and AMG 655 compensate for each

other’s liabilities, and our results support continued investigation

of this combination for cancer therapy.

EXPERIMENTAL PROCEDURES

Cells and Reagents

Tumor cell lines were obtained from established sources (American Type

Culture Collection, Deutsche Sammlung von Mikroorganismen und Zellkultu-

ren, and Sigma) and maintained according to suppliers’ recommendations.

AMG 655 was made as previously described (Kaplan-Lefko et al., 2010).

Mouse antihuman DR5 antibodies M410, M411, M412, M413, and M415

were made at Immunex/Amgen, and M413 has been previously described

(Griffith et al., 1999). LZ-hu Apo2L/TRAIL, huApo2L/TRAIL (114–281),

N297A-AMG 655, AMG 655 Fab, AMG 655 Fab02, DR5 extracellular domain,

and anti-mu DR5 clone MD5-1 were made at Amgen. Additional details are

available in Supplemental Experimental Procedures.

Cancer Cell


Cell Viability and Caspase-3/7 Activity Assays

For tumor cell viability assays, 2 3 104 cells/well were plated in 96-well plates

and treated for 24 hr (human tumor lines) or 48 hr (mouse tumor lines) in dupli-

cate or triplicate with the indicated amounts of human IgG1 (1 mg/ml), Apo2L/

TRAIL, AMG 655, AMG 655 plus protein G (Pierce) (1 mg/ml), Apo2L/TRAIL plus

AMG 655 (1 mg/ml), MD5-1, Apo2L/TRAIL plus MD5-1 (1 mg/ml), or Apo2L/

TRAIL plus hamster IgG (1 mg/ml). As shown in Figure 3A, a fixed concentration

(68 nM) of AMG 655, AMG 655 Fab, AMG 655 Fab02, or control medium was

added, and cell viability was assayed after 6 hr using Cell Titer Glo (Promega)

or ATPLite (Perkin-Elmer) according to the manufacturer’s protocols. Nonma-

lignant cells were treated for 48 or 72 hr. For coculture experiments, indicated

amounts of 293T cells transected with control pCDNA3.1 vector or pAMG21-

hu TRAIL (T95-G281) (generated at Amgen) were mixed with 2 3 104 cells

(SkLu-1, SW1573, SW403) in 96-well plates. After 4 hr, caspase-3/7 activity

was measured using a Caspase-Glo assay (Promega) according to the manu-

facturer’s protocols. Additional details are available in Supplemental Experi-

mental Procedures.

Flow Cytometry

Antibodies used for flow cytometry were as follows: anti-TRAIL: M180; anti-

DR4: M271; anti-DR5: M413 (all at 10 mg/ml, all made at Immunex/Amgen)

with goat antimouse Fab-PE secondary (Jackson Labs); and anti-mDR5:

biotinylated MD5-1 (1.25 mg/ml; eBioscience). After washing, cells were resus-

pended in 10 mg/ml biotinylated secondary for 45 min at 4�C, washed again,

and incubated with 10 mg/ml streptavidin-PE (BD PharMingen) for 45 min at

4�C. For analysis of Apo2L/TRAIL and Apo2L/TRAIL receptors on human pri-

mary tumor cells, tumor-derived cell suspensions and control cell lines were

stained for 30 min at room temperature with antibody cocktails containing

EpCAM-APC (clone EBA-1; BD Biosciences), CD45 (PE-Cy7; clone HI30;

BD Biosciences), and antibodies against IgG1, IgG2b, Apo2L/TRAIL, DR4,

DR5, DcR1, or DcR2. Before flow cytometric analysis, samples were stained

with 5 mg/ml 7-AAD (Life Technologies) to exclude dead and apoptotic cells.

For each sample, 104 viable EpCAM+ events were acquired. Additional details

are available in Supplemental Experimental Procedures.

Gel Filtration Analysis

WM35 lysates (0.75 ml) prepared from 6 3 107 cells for each condition were

applied to a Superose 6 10/300 GL column (GE Healthcare) equilibrated in

25 mM Tris (pH 7.5), 150 mM NaCl, 10 mM 2-ME, 10% glycerol, and 0.1%

Tween 20, and 1 ml fractions were collected starting just prior to the column

void volume. The column was calibrated using gel filtration standards (BioRad

151-1901). Fractions were subsequently analyzed by immunoblotting for the

presence of DR5.

Protein Crystallization, Structure Determination, and Refinement

Crystals of the Apo2L/TRAIL-DR5-AMG 655 Fab ternary complex were ob-

tained in hanging drops with 0.1 M Tris (pH 8.0), 1.0 M LiCl, 0.2 M MnCl2,

and 10% polyethylene glycol 6000. These crystals belong to the space group

P6122 with unit cell parameters of a = b = 152 and c = 613 A. Paratone-N min-

eral oil was used as a cryoprotectant. For data collection, crystals were ori-

ented so that the longest axis (c) was perpendicular to the X-ray beam. Diffrac-

tion data were collected on beamline 21-ID-F at the Advanced Photon Source

and processed and scaled with HKL 2000. The structure of the ternary Apo2L/

TRAIL-DR5-AMG655 Fab complex was solved bymolecular replacement with

PHASER using the structures of the binary Apo2L/TRAIL-DR5 complex (Pro-

tein Data Bank [PDB] accession number 1D0G) and variable (VH and VL) and

constant (CH1 and CL) domains of an IgG1 antibody (PDB accession number

3B2U) with 89% sequence similarity to AMG 655 as the search models. Model

building was carried out with Coot, and refinement was done using Refmac in

CCP4. In the final structure, 99.1% of the residues are in the allowed regions of

the Ramachandran plot. Details on data processing and refinement statistics

are provided in Table 1. Additional details are available in Supplemental Exper-

imental Procedures.

Mouse Efficacy and Tolerability Studies

For all in vivo studies, mice were cared for in accordance to the Guide for the

Care and Use of Laboratory Animals, eighth edition. All research protocols

were approved by the Amgen Institutional Animal Care and Use Committee.

For xenograft models, mice were injected with 2 3 106 SW403 or 5 3 106

H460 tumor cells. Mice were randomized into groups (n = 10) and treated intra-

peritoneally (i.p.) with Apo2L/TRAIL (60 mg/kg, five times per week), AMG 655

(100 mg/mouse, twice per week), or the combination for two cycles. For synge-

neic models, mice were injected with 53 105 Renca or LLC1 cells. Mice were

randomized into groups (n = 10) and treated i.p. with Apo2L/TRAIL (5 mg/kg,

five times per week), MD5-1 (1 mg/mouse, once per week), or the combination

for two cycles. For the tolerability model, mice were treated with PBS, Apo2L/

TRAIL (60 mg/kg, once per day for 5 days), MD5-1 (200 mg, twice per week), or

the combination (n = 10/group). Mice were necropsied on day 5 of treatment.

Additional details are provided in Supplemental Experimental Procedures.

Immunoprecipitation and Western Blotting

SkLu-1 and Colo205 cells treated for 4 hr with control medium, AMG 655,

Apo2L/TRAIL, or Apo2L/TRAIL plus AMG 655 at the indicated amounts and

lysed. Seven microliters of anti-FADD (Sigma F8053), anti-caspase-8 (Cell

Signaling 9746), or IgG (Fisher BP268550) was added to each reaction, and

after 4 hr, 50 ml of 50% protein G Sepharose slurry was added and rotated

at 4�C overnight. For Figure 3B, 50 ml of 50% protein G Sepharose slurry

was added and rotated at 4�C overnight. Immunoprecipitates were analyzed

by immunoblotting with anti-TRAIL (Abcam ab9959), anti-hIgG (Abcam

ab109489), anti-FADD (Cell Signaling 2782), anti-DR5 (Cell Signaling 8074),

and anti-caspase-8 (Cell Signaling 9746) or antitubulin (Sigma T5168), using

the Odyssey image system (LI-COR Biosciences).

Immunofluorescence and Confocal Microscopy

SkLu-1 cells were treated with Apo2L/ TRAIL or AMG 655 alone or in combi-

nation as indicated for 15 min at 37�C. Cells were fixed, permeabilized, and

incubated overnight at 4�C with rabbit anticleaved caspase-8 (Cell Signaling

9496) andCy3-conjugated donkey antihuman IgG (4 mg/ml; Jackson Immunor-

esearch). After washes in Tris-buffered saline and Tween-20, AlexaFluor 488-

conjugated donkey antirabbit IgG (4 mg/ml; Life Technologies) was applied.

After final washes, cells were mounted in ProLong Gold antifade reagent

(Life Technologies) and imaged on a Zeiss LSM 510 confocal microscope.


Immunohistochemistry

All nonhuman primates were cared for in accordance with the Guide for the

Care and Use of Laboratory Animals. Animals were socially housed in a facility

accredited by the Association for Assessment and Accreditation of Laboratory

Animal Care International. All research protocols were approved by the Sierra

Biomedical Institutional Animal Care and Use Committee. For human and

nonhuman primate DR5 staining, mouse antihuman DR5 (M413) (Amgen) at

5 mg/ml for 60min ormouse IgG1 isotype control (BD PharMingen 550878), fol-

lowed by biotinylated goat antimouse IgG (H&L) (Vector Labs, BA-9200) for

30 min, was used. For mDR5 staining, hamster antimouse DR5 (eBioscience

14-5883), or hamster IgG isotype control (eBioscience 14-4888) at 7.5 mg/ml

for 60 min, followed by biotinylated rabbit anti-hamster IgG (Southern

Biotech 6215-05) for 30 min, was used. For caspase-3, cleaved caspase-3

(Cell Signaling 9661) for 60 min, followed by goat antirabbit Envision (Dako)

for 30 min, DAB (Dako) for 5 min, and Mayer’s hematoxylin (Dako) for 30 s

was used. Additional details are provided in Supplemental Experimental

Procedures.

Primary Tumor Disaggregate Analysis

Non-small-cell lung tumor samples were collected under Institutional Review

Board approval for each institution, with appropriate informed consent, from

surgical resections or after biopsy. Freshly resected human tissue samples

were obtained from the following institutions: Asterand USA, Bio-Options,

MT Group, and Conversant Bio. In all cases, tissue obtained was surplus to

standard clinical practice, and patient identity and identifying data were

redacted from tissues and clinical data. Freshly resected lung tumors were di-

gested, and cells were suspended in RPMI. Aqua Viability Reagent (Life Tech-

nologies) was added to the cell suspension per the manufacturer’s protocol to

label dead cells. A fraction of cells were removed for live cell flow cytometry

to measure receptor surface expression. Tumors (106 cells) were incubated

for 18 hr with vehicle, 1 mg/ml human IgG1 (Life Technologies), AMG 655

(1 mg/ml), or Apo2L/TRAIL (0.13, 1.6, or 2 mg/ml) combined with vehicle,


Cancer Cell


1 mg/ml human IgG1, or AMG 655 (1 mg/ml). Cells were fixed, permeabilized,

and stained with fluorochrome-conjugated antibodies specific for cleaved

caspase-3, EpCAM, and pan-cytokeratin. Samples were run on an LSR II using

a gating strategy to identify EpCAM+ and cytokeratin+ events and exclude

Aqua Viability dye-positive events (i.e., debris). Results were reported as the

percentage of viable events remaining in treated samples relative to vehicle.


Statistical Analysis

Experiments were repeated three times unless otherwise indicated. Data were

presented as mean ± SEM. For in vivo tumor studies, statistical analysis

was performed using JMP7 software (SAS Institute). Repeated-measures

ANOVA was performed over time using Dunnett’s post hoc test to determine

the p value.

ACCESSION NUMBER

The PDB code for this structure is 4N90.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

six figures, and one table and can be found with this article online at http://

dx.doi.org/10.1016/j.ccr.2014.04.028.

AUTHOR CONTRIBUTIONS

J.J.K., J.P., T.-H.H., and H.D. performed in vitro cell line work. R.M. and X.Z.

performed the colorectal cancer cell panel screen. T.S., T.L.B., and T.D.

performed mouse in vitro and in vivo studies. T.-H.H. performed biochemical

studies. W.C. performed confocal microscopy analyses. D.G.B. provided

pathology support. J.W.O. supported protein production. J.M.R. performed

tumor disaggregate studies. A.M.L. and X.H. performed structure studies.

J.D.G, P.M.H., and I.N.F. guided the project and contributed to experimental

design and data interpretation. P.M.H. and J.D.G. wrote the manuscript.

ACKNOWLEDGMENTS

We thank T. Polverino, B. Fanslow, R. Kendall, J. Hill, D. Saffran, and R. Loberg

for discussions; R. Kegel and B. Thangaraj for protein supply; A. Jones for cell

panel screening; K. Rohrbach for histology support, and J. Danao for receptor

expression analyses. All authors are current or former employees of Amgen

and have received stock or stock options from Amgen.

Received: January 2, 2014

Revised: February 28, 2014

Accepted: April 30, 2014

Published: July 17, 2014

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apo2l/trail and the death receptor 5 agonist antibody amg 655 cooperate to promote receptor...

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