rg7386, a novel tetravalent fap-dr5 antibody,...

Large Molecule Therapeutics

RG7386, a Novel Tetravalent FAP-DR5 Antibody,Effectively Triggers FAP-Dependent,Avidity-Driven DR5 Hyperclustering andTumor Cell ApoptosisPeter Br€unker1, Katharina Wartha2, Thomas Friess2, Sandra Grau-Richards1,Inja Waldhauer1, Claudia Ferrara Koller1, Barbara Weiser2, Meher Majety2,Valeria Runza2, Huifeng Niu3, Kathryn Packman3, Ningping Feng3,Sherif Daouti3, Ralf J. Hosse1, Ekkehard M€ossner1, Thomas G.Weber2,Frank Herting2,Werner Scheuer2, Hadassah Sade2, Cuiying Shao4,Bin Liu4, Peng Wang4, Gary Xu4, Suzana Vega-Harring2, Christian Klein1,Klaus Bosslet2, and Pablo Uma~na1

Abstract

Dysregulated cellular apoptosis and resistance to cell death arehallmarks of neoplastic initiation and disease progression. There-fore, the development of agents that overcome apoptosis dysre-gulation in tumor cells is an attractive therapeutic approach.Activation of the extrinsic apoptotic pathway is strongly depen-dent on death receptor (DR) hyperclustering on the cell surface.However, strategies to activate DR5 or DR4 through agonisticantibodies have had only limited clinical success. To pursuean alternative approach for tumor-targeted induction of apopto-sis, we engineered a bispecific antibody (BsAb), which simulta-neously targets fibroblast-activation protein (FAP) on cancer-associated fibroblasts in tumor stroma and DR5 on tumor cells.Wehypothesized that bivalent binding tobothFAP andDR5 leadsto avidity-driven hyperclustering of DR5 and subsequently stronginduction of apoptosis in tumor cells but not in normal cells.

Here, we show that RG7386, an optimized FAP-DR5 BsAb,triggers potent tumor cell apoptosis in vitro and in vivo in preclin-ical tumor models with FAP-positive stroma. RG7386 antitumorefficacywas strictly FAP dependent, was independent of FcR cross-linking, and was superior to conventional DR5 antibodies. Incombination with irinotecan or doxorubicin, FAP-DR5 treatmentresulted in substantial tumor regression in patient-derived xeno-graft models. FAP-DR5 also demonstrated single-agent activityagainst FAP-expressing malignant cells, due to cross-bindingof FAP and DR5 across tumor cells. Taken together, these datademonstrate that RG7386, a novel and potent antitumor agentin bothmono- and combination therapies, overcomes limitationsof previous DR5 antibodies and represents a promisingapproach to conquer tumor-associated resistance to apoptosis.Mol Cancer Ther; 15(5); 946–57. �2016 AACR.

Introduction

Programmed cell death (apoptosis) is a crucial process fornormal development, tissue homeostasis, and the immuneresponse in multicellular eukaryotic organisms. Impaired apo-ptosis, in turn, plays a key role in cancer pathogenesis andcontributes to poor chemotherapy responses. Thus, development

of agents that restore/activate apoptotic pathways specifically intumor cells has emerged as an important therapeutic strategy. Theextrinsic apoptotic pathway is triggered by external signals,including proapoptotic FAS-ligand and TRAIL, which bind to andactivate cell surface-anchored death receptors (DR) such as FAS,DR4, or DR5 through receptor oligomerization. DR activationpromotes the formation of the death-inducing signaling complex,

1Roche Pharma Research and Early Development, Roche InnovationCenter Zurich, Schlieren, Switzerland. 2Roche Pharma Researchand Early Development, Roche Innovation Center Munich, Penzberg,Germany. 3Roche Pharma Research and Early Development, RocheInnovation Center New York, New York, New York. 4Roche PharmaResearch and Early Development, Roche Innovation Center Shanghai,Shanghai, China.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

P. Br€unker and K. Wartha contributed equally to this article.

Current address forK.Wartha: Actelion Pharmaceuticals, Allschwill, Switzerland;current address for H. Niu: Takeda Pharmaceuticals International Co., Cam-bridge, MA; current address for K. Packman: Janssen, Pharmaceutical Compa-

nies of Johnson and Johnson, Boston, MA; current address for N. Feng: MDAnderson Cancer Center, Institute for Applied Cancer Science, Houston, TX;current address for S. Daouti: Bristol-Myers Squibb, Lawrence Township, NJ;current address for T.G. Weber: Department of Radiology, University of Cali-fornia, San Diego, La Jolla, CA; and current address for P. Wang: CARsgenTherapeutics, Shanghai, China.

Corresponding Author: Pablo Uma~na, Roche Innovation Center Zurich,Wagistrasse 18, CH-8952 Schlieren, Switzerland. Phone: 414-4755-6134;Fax: 414-4755-6160; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0647

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 15(5) May 2016946

on September 12, 2018. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst April 1, 2016; DOI: 10.1158/1535-7163.MCT-15-0647

http://mct.aacrjournals.org/

leading to subsequent activation of caspases 3, 6, and 7 and cellapoptosis (1, 2). Several proapoptotic receptor agonists (PARA),including mAbs, have been developed to target this extrinsicapoptotic pathway (3, 4). However, despite promising preclinicaldata, none of these approaches have thus far demonstratedsignificant clinical efficacy (5–7). Although some antibodiesappear to be able to autonomously activate DR4 and DR5, manyrequire additional cross-linking to achieve optimal activity in vitro(8–11). In vivo, and as demonstrated for drozitumab, a fullyhuman DR5 agonistic antibody (8), this cross-linking activity islikely provided by activatory or inhibitory Fcg receptors (FcgR) onleukocytes in the tumor microenvironment (12, 13). Low orvariable infiltration of FcgR-expressing leukocytes, reduced target-ing of DR agonistic antibodies due to systemic binding to DRs onnontumor cells or to FcRs on circulating cells, and/or competitionfor FcRs by high levels of endogenous immunoglobulins, mayaccount for the lack of clinical efficacy observed for these agonisticantibodies.

Fibroblast-activation protein (FAP) is a homodimeric, single-pass type II membrane protein expressed in reactive stromalfibroblasts of more than 90% of epithelial malignancies,including breast, colorectal, and lung cancers and on malignantmesenchymal cells of bone and soft tissue sarcomas (14–16).Here, we exploited FAP expression on the tumor stroma todevelop novel bispecific antibodies (BsAb) consisting of a DR5agonist and anti-FAP antibody fusion to enhance targeteddelivery of the DR5 agonist, induce avidity-driven DR5 hyper-clustering and activate the extrinsic apoptotic pathway specif-ically in tumor cells. We demonstrate that FAP-DR5 antibodiessignificantly enhance tumor cell apoptosis in vitro across severaltumor indications compared with the parental drozitumabmAb. Importantly, the antitumor activity of the FAP-DR5antibodies was dependent on the presence of FAP-expressingfibroblasts. Using an optimized FAP-DR5 BsAb RG7386, wefurther show that the mode of action of the DR5 agonist can beuncoupled from FcgR-dependent cross-linking when fused tothe FAP-binding moiety. RG7386 demonstrates in vivo efficacyin several xenograft models, including patient-derived xeno-graft (PDX) models with FAP expression either on stroma or onmalignant cells. Finally, RG7386 in combination with irinote-can or doxorubicin enhances the antitumor effects of therespective monotherapies in vivo and results in complete tumorremission.

Materials and MethodsCell lines and host strains

HEK293 EBNA, NIH-3T3, MDA-MB-231, DLD-1, Colo205,and LOX-IMVI were obtained from LGC Standards (Europeandistributor for the ATCC), A549 fromECACC,GM05389 from theCoriell Institute for Medical Research (Camden, NJ), and 1833-PPOP233 from Promega Corp. All cell lines were purchasedbetween 2007 and 2010 and authenticated by morphologic andgrowth curve analysis. For more details see Supplementary Mate-rials and Methods.

Vector constructionThe construction of expression vectors for standard IgGs

(immunoglobulin G) and BsAbs was performed according tostandard recombinant DNA technologies. Each antibody chaincDNA was amplified by PCR or generated by conventional gene

synthesis at Geneart and transiently expressed in HEK293 EBNAcells under the MPSV promoter (myeloproliferative sarcomavirus). Bispecific FAP-DR5 antibodies were generated by fusionof a FAP-binding domain to the DR5 IgG heavy chain at the C-terminus via a (G4S)4 linker. The DR5 portion consisted of thevariable-light chain (VL) and variable-heavy chain (VH) of dro-zitumab (described by Adams in US2007/003141401 A1) ornovel DR5 antibodies generated by phage display. To minimizelight-chain mispairing side-products, we used CrossMab technol-ogy (17). The FAP-binding unit was engineered as a crossed Fab inwhich the VH was fused to the constant-light chain (CL) and theVL to a CH1 (constant-heavy 1) domain. To prevent FcR binding,three mutations in the Fc region [PGLALA: P329G (patent No.WO 2012130831) and L234A and L235A (18)] were generatedby standard site-directed mutagenesis in the indicated BsAbs.All amplified or synthesized genes were confirmed by DNAsequencing.

Production and characterization of anti-DR5 bindersAntibodies with specificity for human DR5 were selected

from a generic phage-display antibody library in the Fabformat. Specific binders were identified by ELISA assays andclones exhibiting signals on human DR5 but none on humanDCR2 and human IgG1 were short-listed for further analyses.All BsAbs and antigens (if not obtained from a commercialsource) were transiently produced in HEK293 EBNA cells andpurified using protein A affinity purification and size exclusionchromatography. The purity and molecular weight of the BsAbpreparations were determined by Microchip capillary electro-phoresis (CE)-SDS on Caliper's LabChip GXII using the ProteinExpress LabChip Kit. For more details see SupplementaryMaterials and Methods.

Binding of BsAbs and flow cytometrySimultaneous binding of BsAbs to human DR5 as well as hu or

muFAP was assessed by surface plasmon resonance (SPR) usingBiacore T100. The binding of BsAbs on cells was determined usinga time-resolved fluorescence resonance energy transfer (TR-FRET)assay. In the flow cytometry, anti-FAP and anti-DR5were used as aprimary and goat anti-human Fc specific PE-labeled as a second-ary antibody. For more details see Supplementary Materials andMethods.

Apoptosis detection by DNA fragmentationGM05389 fibroblasts (1 � 104 cells/well), harvested with

Cell Dissociation Buffer and resuspended in appropriatemedium, were plated overnight at 37�C in 96-well plates.Fibroblasts were then incubated with antibodies (anti-DR5IgGs or FAP-DR5 BsAbs) alone or together in a 1:1 ratio with agoat anti-human IgG cross-linking antibody (Sigma #I2136)at the indicated concentrations for 10 minutes. Tumor cells(1 � 104 cells/well) were harvested in Cell Dissociation Buffer,resuspended in appropriate medium and were added to thefibroblasts. Cells were incubated with the antibodies for24 hours at 37�C followed by centrifugation for 10 minutesat 200 � g at room temperature. The amount of fragmentedDNA was determined using Cell Death detection ELISAPLUS

(Roche Applied Science #11 774 425 001) according to themanufacturer's instructions. Where indicated Vmax values werecalculated from absorbance readings taken every minute

FAP-Dependent DR5 Hyperclustering Drives Tumor Cell Death

www.aacrjournals.org Mol Cancer Ther; 15(5) May 2016 947




Figure 1.FAP-DR5 BsAb and apoptosis activity. A, principle of tumor-targeted apoptosis by BsAbs consisting of a CrossFab anti-FAP unit (mAb007 or mAb082)fused to the C-terminus of the drozitumab heavy chain using a 20mer GS linker. The mAb007 anti-FAP moiety was fused to drozitumab heavy chainin either a VHCL or VLCH1 configuration. A nontargeting DP47GS_drozitumab antibody was generated in the VHCL format (not shown here). Atrivalent mAb007_drozitumab molecule (1þ2; VHCL) was also developed. (Continued on the following page.)

Br€unker et al.

Mol Cancer Ther; 15(5) May 2016 Molecular Cancer Therapeutics948




over 20 minutes at 405 nm using a Versamax MicroplateReader (Molecular Devices). Measurements at 490 nm wereused as a reference. In other experiments, end-point absor-bance readings were taken at 405 nm using a Tecan SpectraRainbow Reader.

Mouse xenograft modelsAll experiments were conducted according to the guidelines

of the German Animal Welfare Act (GV-Solas; Felasa; TierschG).For the cell line–based xenograft models, tumor cells (2 �

106 cells) were co-injected s.c. onto Balb/c nude mice (CharlesRiver Laboratories) together with murine fibroblasts (NIH3T3,0.4 � 106 cells). Cell inoculation was supplemented withMatrigel (Basement Membrane Matrix, BD Biosciences). Thefragment-based colorectal cancer xenograft Co5896 and sarco-ma tumor xenograft Sarc4605 originally obtained from patientswere passaged approximately three to five times until estab-lishment of stable growth patterns. For the subsequent in vivoPDX studies, tumors were removed from xenografts in serialpassage in donor nude mice, cut into fragments (4–5 mmdiameter), and placed in PBS until subcutaneous implantationin recipient mice.

Mice (n ¼ 10/group) were subjected to stratified random-ization based on primary tumor size (median tumor volume ofapproximately 100–150 mm3) before treatment. Unless oth-erwise indicated, FAP-DR5 BsAb RG7386, drozitumab anddrozitumab-PGLALA were administered once weekly i.v. Iri-notecan (15 mg/kg) was administered intraperitoneally, doxo-rubicin (5 mg/kg) was given weekly intravenously, and salinewas used as the vehicle control. Tumor volume was measuredby caliper twice weekly [(length � width2)/2] and the percent-age of tumor growth inhibition (TGI) relative to controlanimals was calculated. All treatments were well tolerated asindicated by stable body weights throughout the duration ofthe studies.

Bioluminescence detection of apoptosis in vivo1833-PPOP233 cells (4 � 106 cells/mouse) and NIH3T3 cells

(1 � 106 cells/mouse) were subcutaneously coinjected into theright flank of 6- to 8-week-old Scid/beige mice (Charles RiverLaboratories) with body weights of 18 to 24 g. After treatmentwith BsAbs, mice were injected with a single dose of D-luciferin(Promega; 150 mg/kg in PBS; i.p.) 10 minutes before biolumi-nescence imaging using IVIS spectrum (Caliper Life Sciences). Theacquired bioluminescence signals in a region of interest (ROI)over the tumor site were measured as average radiance (p/sec/cm3/sr) with Living Image software (Caliper Life Sciences). EveryROI signal was divided by the appropriate baseline ROI signal tocalculate fold induction (19, 20).

IHCIHC protocols for cleaved caspase-3 and FAP were performed

on Discovery or BenchMark XT automated strainers. All

reagents except the antibodies were obtained from VentanaMedical Systems. For more details see Supplementary Materialsand Methods.

Statistical analysesNonparametric treatment-to-control-ratios (TCR) based on

endpoint analysis and the two-sided nonparametric confidenceintervals (CI) compared with the vehicle group were calculatedto assess statistical significance in in vivo studies. A TCR valueof 0 indicates complete tumor regression and CIs of less than1 represent statistically significant results. Relative TGI was cal-culated according to the formula (1�[(T�T0)/(C�C0)]) � 100.

ResultsA novel bispecific anti-FAP and anti-DR5 antibody exhibitsdual specificity

To overcome the disadvantages of current DR5 antibodies,and to boost antitumor efficacy, we engineered novel BsAbs thatsimultaneously bind to DR5 on tumor cells and to FAP on thetumor stroma to induce localized DR5 hyper–cross-linking. TheBsAbs consisted of a human anti-DR5 agonist (drozitumab-derived huIgG1) with a C-terminal fusion of an anti-FAP bind-ing moiety (mAb007 or affinity-matured mAb082 clone;Fig. 1A). For heterodimerization of this trivalent molecule, the"knobs-into-holes" technology (21) was implemented. All FAP-drozitumab antibodies exhibited similar binding to humanDR5 as the parental drozitumab and a nontargeting BsAb(drozitumab fused to non-binding DP47GS Cross Fab as acontrol), demonstrating that the process of antibody fusionhad not impaired antibody binding to the DR5 antigen in cells(Fig. 1B; Supplementary Table S1). Interestingly, we found thatthe Fab configuration used to fuse FAP to drozitumab wasimportant for preserving BsAb interaction with the FAP antigen.In a VLCH1 format, mAb007_drozitumab demonstratedreduced binding to FAP (>9-fold), compared with parentalmAb007 antibody. Binding to FAP was similarly reduced bya BsAb with a monovalent FAP-binding domain (Fig. 1C;Supplementary Table S1), corresponding to a loss in avidity.In contrast, in the VHCL format, mAb007_drozitumab andmAb082_drozitumab demonstrated similar binding to FAP(Ki ¼ 0.32 nmol/L and Ki ¼ 0.53 nmol/L, respectively) thatwas comparable with the parental antibodies (Fig. 1C; Supple-mentary Table S1). These data highlight that the VHCL Fabconfiguration is the most suitable format for generating BsAbs.

Importantly, SPR experiments confirmed simultaneous bind-ing of the FAP-drozitumab antibodies to both DR5 and FAPrecombinant proteins, indicative of a fully functioning dualantibody (Supplementary Fig. S1A).

FAP-DR5 bispecific molecules induce tumor cell apoptosisin vitro in the presence of FAP-expressing fibroblasts

To investigate the antitumor efficacy and specificity ofthe FAP-drozitumab BsAbs, tumor cell apoptosis (DNA

(Continued.) B and C, BsAb binding to antigens DR5 (B) and FAP (C) was assessed using a TagLite competition assay in HEK EBNA cells expressinghuDR5 and huFAP extracellular domains, respectively. D–G, analysis of MDA-MB-231 tumor cell apoptosis in coculture with FAP-expressing GM05389 inresponse to drozitumab alone (D), drozitumab þ anti-Fc (cross-linked drozitumab; E) and BsAbs (F–I) at the indicated concentrations. Apoptosis wasdetermined as a measure of DNA fragmentation in an ELISA assay. Vmax values calculated from kinetic measurements taken at 405 nm are shown(mean � SEM; n ¼ 3 independent experiments). See also Supplementary Fig. S1.






fragmentation) was monitored in the presence or absence ofthe FAP-positive human GM05389 fibroblast cell line (Supple-mentary Fig. S1B).

In the absence of FAP-expressing fibroblasts, low concentra-tions of drozitumab or BsAb resulted in minimal MDA-MB-231breast cancer cell apoptosis. However, DNA fragmentation wasobserved at the highest antibody concentrations used (7 and 35nmol/L; Fig. 1D–I). Induction of drozitumab cross-linking byaddition of a secondary anti-human Fc antibody promoted dose-dependent tumor cell apoptosis with a significant 100-fold reduc-tion in EC50 values from 15.2 nmol/L (drozitumab alone) to 0.14nmol/L (cross-linked drozitumab; Fig. 1D and E; SupplementaryTable S2), demonstrating that cross-linking is critical for antibodyefficacy.

In the presence of FAP-expressing fibroblasts, all BsAbssubstantially increased tumor cell apoptosis (Fig. 1F–I). Underthese conditions, BsAb treatment resulted in dose-dependentcell death at concentrations as low as 0.0112 nmol/L withmaximum activity at 7 nmol/L. In addition, the EC50 value ofthe bispecific molecules was approximately 20-fold lower ascompared with the anti-Fc cross-linked drozitumab (0.014 vs.0.33 nmol/L; Supplementary Table S2). Importantly, the paren-tal drozitumab antibody did not trigger apoptosis in the sameMDA-MB-231 and fibroblast coculture assay without cross-linking. These data clearly implicate the requirement for FAPexpression in the tumor stroma for bispecific FAP-drozitumabantibody function. Interestingly, the antibody configurationdid not appear to hinder the in vitro potency of the bispecificmolecules (Fig. 1G and H; Supplementary Table S2) despitedifferential binding to the FAP antigen (Fig. 1B and C). How-ever, it appeared that antibody valence for the FAP antigen wascrucial for BsAb potency (EC50 value increased from 0.01 in thebivalent molecule to 0.57 nmol/L in the monovalent format;Supplementary Table S2). Altogether, the BsAb containing themAb007 anti–FAP-binding moiety appeared to be less specificand induced tumor cell apoptosis in the absence of fibroblastsat lower concentrations. Therefore, the mAb082 anti–FAP-binding moiety was adopted for further BsAb developmentand in vivo experiments.

The FAP dependency of mAb082_drozitumab to trigger DR5clustering and apoptosis was further evaluated in the colorectalcancer DLD-1 cell line. In the presence of NIH3T3 fibroblaststhat lack FAP expression in vitro (Supplementary Fig. S1B),the antibody failed to trigger tumor cell apoptosis (Supple-mentary Fig. S1C). In contrast, stable expression of murine FAPin NIH3T3 cells (muFAP NIH3T3; Supplementary Fig. S1B)resulted in significant antibody-mediated tumor cell death(Supplementary Fig. S1D). Moreover, the untargeted controlmolecule DP47GS_drozitumab, which does not bind FAP,failed to induce apoptosis even in the presence of FAP-expres-sing cells (Supplementary Fig. S1C–S1D). Together, these datademonstrate the fundamental requirement for FAP-positivecells in cross-linking of the FAP-drozitumab BsAbs and in theactivation of the DR5 apoptotic pathway.

BsAb-mediated DR5 cross-linking reveals superior apoptosisinduction over untargeted drozitumab in a variety of tumorcell lines

To assess the generalizability of the FAP-drozitumab anti-body, five tumor cell lines were evaluated for DR5 cell surfaceexpression and apoptosis sensitivity in response to BsAb treat-

ment. Flow-cytometry analyses revealed two cell lines with high(ACHN; renal carcinoma and DLD-1 colorectal cancer), twowith moderate (Colo205; colorectal cancer and MDA-MB-231breast cancer), and one cell line with low DR5 expression(A549; lung carcinoma; Fig. 2A). All of these tumor cell linesdemonstrated susceptibility to FAP-drozitumab antibody-mediated apoptosis (Fig. 2B) in a dose-dependent manner.Moreover, BsAb action required FAP targeting as the untargetedBsAb control (DP47GS_drozitumab) failed to induce apoptosisat low doses (Fig. 2B). As previously discussed, the apoptoticactivity of DP47GS_drozitumab at higher concentrations couldbe attributed to the action of drozitumab alone (Fig. 1D).Intriguingly, and in agreement with a previous publication(22), no obvious correlation was apparent between DR5 cellsurface expression and FAP-drozitumab BsAb-induced apopto-sis (Fig. 2; Supplementary Table S3).

Altogether, these data indicate the potential for the broadapplication of FAP-drozitumab BsAbs in a range of tumorindications and highlight the importance of FAP targeting forBsAb function.

An optimized bispecific FAP-DR5 antibody, RG7386 inducesapoptosis in vitro and inhibits in vivo tumor growth in xenograftmouse tumor models

Conventional anti-DR5 antibodies such as drozitumab relyon FcgR-dependent mechanisms to achieve DR5 clustering(13). Therefore, efficient DR5 activation may be hindered byseveral factors, including high concentrations of endogenousIgG, present in patient sera, and by the activity of FcgR-expres-sing immune cells within normal tissue leading to limitedantitumor efficacy and unwanted side-effects. In addition,FcgR-dependent cross-linking in the liver has been shown tomediate hepatotoxicity for murine FAS agonistic antibodies(23), and a similar mechanism may play a role in the hepaticside-effects reported for previous DR5 agonists in the clinic(24). Moreover, our data suggested that the parental drozitu-mab antibody triggers cross-linker–independent cytotoxicityat concentrations above 1 nmol/L (Fig. 1D). To eliminatethese confounding variables and to improve future clinicalsafety profiles, we generated a BsAb composed of another DR5antibody mAb096, which does not trigger apoptosis in theabsence of cross-linking (Supplementary Fig. S2A). Further-more, to prevent interaction with host FcgR on host immunecells, we introduced previously reported IgG1 point mutations[PGLALA: P329G (patent No. WO 2012130831) and L234Aand L235A (18)] in mAb096 that completely abolish FcgRand C1q binding while still retaining cross-linking activityby anti-Fc antibodies.

In the absence of fibroblasts, this optimized FAP-DR5 BsAb(mAb096-mAb082 in VHCL configuration) named RG7386(characterized in Supplementary Table S4 and SupplementaryFig. S2B), promoted MDA-MB-231 tumor cell apoptosis onlywith the addition of a secondary anti-Fc antibody similarlyto the parent mAb096–anti-Fc combination (Fig. 3A). Impor-tantly, the BsAb displayed more potent tumor cell apoptosis inthe presence of FAP-expressing GM05389 fibroblasts (EC50 ¼0.3 nmol/L) compared with the cross-linked parent molecule(EC50 ¼ 5.3 nmol/L) and the addition of the anti-Fc secondaryantibody had no additive effects on RG7386-mediated MDA-MB-231 tumor cell death in cocultures (EC50 ¼ 0.5 nmol/L;Fig. 3B). In addition, RG7386 had minimal effects on fibroblast

Br€unker et al.





cell survival in monocultures (Fig. 3C). These data demonstratethe strong and specific antitumor effects of RG7386 in responseto FAP-mediated cross-linking.

The antitumor efficacy of RG7386 was further investigated invivo in xenograft mouse models. To obtain comparable FAPexpression levels with those observed in the human tumor

stroma, FAP-expressing NIH3T3 cells were s.c. coinjected withDLD-1 tumor cells onto nude mice and FAP expression in thetumor microenvironment was confirmed with IHC (Fig. 4A).Dose-optimization studies revealed effective TGI followingonce weekly administration of RG7386 at 3 and 10 mg/kgrelative to vehicle control (Supplementary Fig. S3A). In terms

Figure 2.FAP-drozitumab antibody induces apoptosis in several tumor cell lines. A, flow-cytometry analysis of DR5 cell surface expression, using drozitumab IgG, incell lines representing different tumor indications. B, analysis of tumor cell apoptosis in coculture with FAP-expressing GM05389 fibroblasts in response tomAb082_drozitumab or DP47GS_drozitumab nontargeting control. Apoptosis was determined as a measure of DNA fragmentation in an ELISA assay. Vmax valueswere calculated from kinetic measurements taken at 405 nm and represented as fold induction over untreated cells. Dose–response curves (mean � SEM; n ¼ 3independent experiments) are shown.






of pharmacokinetics, the exposure to RG7386 (AUC0–72h)appeared to increase in a dose-dependent manner with expo-sure of the largest dose (10 mg/kg) being somewhat higher thanexpected (Supplementary Fig. S3B).

Importantly, treatment with RG7386 (10 mg/kg) significant-ly inhibited tumor growth (TCR, 0.31; 95% CI, 0.17–0.66; TGI¼ 89%) whereas drozitumab-PGLALA (mutant drozitumabunable to be cross-linked without addition of a secondaryanti-Fc antibody), failed to have any effect on tumor size ascompared with vehicle-treated mice (TCR, 1.075; 95% CI,0.65–2.43; TGI ¼ 22%; Fig. 4A). As RG7386 also lacks theseFcgR-binding sites, these data strengthen the notion that FAP-dependent cross-linking drives the antitumor effects of thisFAP-DR5 BsAb. Importantly, the in vivo efficacy of the FAP-

DR5 antibody complex to inhibit tumor growth was recapitu-lated using the previous mAb082_drozitumab molecule in thesame DLD-1 colorectal cancer model (TCR, 0.12; 95% CI, 0.00–0.33; TGI > 100% compared with vehicle control) and in theMDA-MB-231 breast cancer model (TCR, 0.34; 95% CI, 0.24–0.47; TGI: 77% compared with vehicle control; SupplementaryFig. S4), demonstrating the potential broad application of thesebispecific molecules. Moreover, drozitumab coupled to thenontargeting DP47GS molecule did not affect tumor growth,further confirming the FAP-dependent targeting action of theFAP-DR5 antibodies to inhibit tumor growth in vivo (Supple-mentary Fig. S4).

To determine whether the RG7386-mediated suppression oftumor growth was specifically a result of DR5-activated tumorcell apoptosis and not through an unidentified mode of anti-body action, cell apoptosis was monitored noninvasively in ahuman tumor xenograft mouse model by bioluminescenceimaging. The 1833-PPOP233 cells (human bone-metastasizingMDA-MB-231 subclone) expressing a split-luciferase constructwith a caspase 3/7 cleavage site (20), together with FAP-expres-sing NIH3T3 cells were s.c. inoculated in mice. Cleavage ofthe luciferase construct, resulting in a detectable luminescencesignal, was assessed over time and used as a measure ofapoptosis. RG7386 treatment (1.0 and 10 mg/kg) of tumor-bearing mice resulted in a rapid and significant increase in theluminescence signal indicative of apoptosis induction down-stream of activated caspase-3/7 (Fig. 4B). Importantly, theenhanced luminescence signal denoting increased apoptosis isin agreement with inhibition of tumor growth, in the samemouse model, following RG3786 treatment (Fig. 4C). Increas-ed caspase-3 activation (brown staining) was also confirmedin the vicinity of stromal bands by IHC staining of RG7386-treated DLD-1 / 3T3 coinjected tumor sections (Fig. 4D). Thesedata clearly demonstrate that RG7386 induces tumor cellapoptosis in vivo to restrict tumor growth.

The bispecific FAP-DR5 antibody RG7386 demonstrates in vivoefficacy in PDX models and more than additive effects incombination with irinotecan

To further validate the in vivo efficacy of RG7386 in a morerelevant disease background, a patient-derived fragment-basedcolorectal cancer tumor (Co5896) was s.c. transplanted ontoNMRI nude mice and FAP expression was confirmed with IHCanalysis (Fig. 5A). RG7386 treatment (30mg/kg, twice weekly) ofthese mice led to a significant decrease in tumor burden (TCR,0.412; 95% CI, 0.24–0.84; TGI ¼ 76%; Fig. 5B). Interestingly,comparison of treatment with control groups revealed that acombination of RG7386 and the standard-of-care irinotecan(chemotherapeutic agent) translated into superior efficacy (TCR,0.00; 95%CI, 0.00–0.004; TGI > 100%) relative to either RG7386(TCR, 0.19; 95%CI, 0.05–0.37) or irinotecanmonotherapy (TCR,0.036; 95% CI, 0.02–0.09) alone. Direct comparison betweeneach treatment group further confirmed that the enhanced efficacyof RG7386 and irinotecan combination was more than additive(TCR, 0.00; 95% CI, 0.00–0.019, and TCR, 0.00; 95% CI, 0.00–0.017, compared with RG7386 and irinotecan monotherapies,respectively) and resulted in prolonged antitumor activity withcomplete eradication of tumors in all mice (Fig. 5C). Together,these data demonstrate the potential cumulative effects ofRG7386 when used with the standard-of-care for the treatmentof human colorectal cancers.

Figure 3.RG7386 induces tumor cell apoptosis in invitro cocultures. A andB, analysis ofMDA-MB-231 cell apoptosis in monocultures (A) and in coculture withFAP-expressing GM05389 fibroblasts (B) in response to the indicatedtreatments. C, analysis of RG7368-mediated apoptotic effects inGM50389 monocultures. Apoptosis was determined as a measure of DNAfragmentation in an ELISA assay. Endpoint absorbance measurements at405 nm are shown (representative experiment of n ¼ 3–5). See alsoSupplementary Fig. S2.

Br€unker et al.





RG7386 demonstrates significant efficacy in FAP-positivehuman mesenchymal tumor models in mice

Thus far, we have focused on the role of RG7386 in initiatingtumor cell apoptosis with a complete dependence on thepresence of FAP-positive fibroblasts. However, FAP expressionis not restricted to the tumor stroma and can be found as aminor or major component of malignant cells in mesenchymal-derived tumors. To investigate whether RG7386 could be usedto directly target FAP-positive tumors, LOX-IMVI desmoplasticmelanoma cells were s.c. injected onto mice and tumors were

allowed to develop. Treatment with 10 mg/kg of RG7386triggered a significant inhibition of tumor growth comparedwith vehicle control (TCR, 0.073; 95% CI, 0.03–0.32) andcompared with drozitumab-PGLALA (TCR, 0.27; 95% CI,0.17–0.48), indicating that FAP-dependent cross-linking con-tributes to enhanced efficacy (Fig. 6A). Moreover, RG7386,administered as a single agent, substantially restricted tumordevelopment (TCR, 0.16; 95% CI, 0.06–0.36; TGI, 99%) in anosteosarcoma Sarc4605 fragment-based PDX model with 100%FAP expression (Fig. 6B). In the same model, complete tumor

Figure 4.RG7386 induces tumor cell apoptosis in vivo in human xenograft mouse models. A, antitumor efficacy of RG7386 in a mouse xenograft model. Nude micewere subcutaneously coinjected with human DLD-1 and NIH3T3 cells and subjected to RG7386 (twice weekly doses) or Drozitumab_PGLALA antibodytreatment when tumor volume was approximately 100 mm3 in size. Shown is a representative FAP IHC of the tumor (median tumor volume andinterquartile range (IQR); n ¼ 10 animals/group). B and C, apoptosis was monitored in mice coinjected with bone-metastasizing MDA-MB-231 1833-PPOP233tumor cells and NIH3T3 cells. B, in vivo analysis of apoptosis using a luminescence caspase 3/7 activation reporter assay in response to the indicatedtreatments over 72 hours. Luminescence images were taken at 0, 6, and 72 hours posttreatment. Representative luminescence images at 6 hours are shown(mean � SEM; n ¼ 5 animals/group). C, analysis of TGI in the same model in response to RG7386 (median tumor volume and IQR; n ¼ 10 animals/group).D, IHC validation of caspase-3 activation in response to RG7386. See also Supplementary Fig. S4.






remission (TGI > 100%) was achieved when RG7386 wascombined with the standard-of-care doxorubicin (5 mg/kg;TCR, 0.03; 95% CI, 0.007–0.09; TCR, 0.15; 95% CI, 0.04–0.54 and TCR, 0.06; 95% CI, 0.02–0.14 compared with vehicle,RG7386, and doxorubicin as single agents, respectively;Fig. 6C). Altogether, these data support the use of RG7386 notonly in the treatment of multiple carcinomas associated with aFAP-positive stroma but also as a potential viable therapy formesenchymal tumors directly expressing FAP.

DiscussionMany cancer therapeutic approaches target the specific mole-

cules/signaling pathways that promote the hyperproliferative/antiapoptotic states in tumor cells. The continuing challenge inthese methodologies, as opposed to conventional chemothera-peutics that target all rapidly dividing cells, is to be able tospecifically discriminate between tumor cells and their healthycounterparts without loss in efficacy. For conventional DR5agonistic antibodies, such as drozitumab and conatumumab,efficacy is strongly dependent on cross-linking that is mediatedby FcgRs in in vivo mouse models (11, 13, 25). However, despitevery convincing and potent in vitro and in vivo preclinical data, thismode of agonistic antibody action has severe drawbacks that mayexplain the limited clinical efficacy observed for first-generationDR agonists (5, 26–28).

In mouse xenograft models, significant tumoricidal activityof DR5 agonists is dependent on sufficient infiltration of FcgR-expressing immune cells. However, this situation does notappear to be relevant in humans, where FcgR-mediated anti-body cross-linking may be reduced by physiologic IgG levels.On the other hand, potential adverse events such as elevatedhepatotoxicity are related to nontumor specific, systemic cross-linking of agonistic antibodies via FcgRIIb molecules presentin the liver (23). Consequently, second-generation DR ago-nistic antibodies are currently under development with a focuson both DR cross-linking to enhance efficacy, and on reducingthe risk of toxicity due to non–tumor-targeted effects. Con-atumumab-coated nanoparticles encasing a cytotoxic drug thatallow a sufficient density of antibody paratopes at the tumor

site to activate DR5 are currently under investigation (29).APG350 is a hexavalent scTRAIL–RBD (single-chain TRAILreceptor–binding domain), which in contrast with previousagonistic antibodies triggers multimer formation indepen-dently of FcgRs (30).

In addition to full-length antibodies, different scaffolds suchas atrimers (31) or multivalent monobodies (32, 33) are underevaluation to target DRs. However, the lack of clinical efficacyobserved with untargeted DR5 agonistic antibodies illustratesthat the activity of these molecules in vitro may not necessarilytranslate into clinical efficacy. Multivalent DR5 binders mightprovide a more potent effect as compared with agonisticantibodies, but these also bear the risk of toxic side effects.TAS266, a novel tetravalent and highly potent DR5 agonisticNanobody, was recently entered into a phase I clinical trial, butwas deemed unsafe for human administration due to acutehepatotoxicity (34) that may have been exacerbated by anabsence of a tumor-targeting entity. The DR ligand, TRAIL,untargeted, targeted, or in multimeric formats (35–38) alsohas some disadvantages such as a short half-life and potentialtoxicity, if oligomerized. In addition, as TRAIL does not onlybind to DR4 and DR5, but also to decoy receptors DCR1,DCR2, and OPG (osteoprotegerin; ref.39) in vivo tumor activitymay be limited.

In our study, we eliminated FcgRs-dependent formation ofantibody multimers by introducing three mutations (PGLALA)in the Fc portion of a novel DR5 agonist (mAb096) thatabolish FcgR binding. In addition, we fused the DR5 agonisticantibody to an anti–FAP-bindingmoiety to take advantage of FAPexpression in the tumor stroma as an alternative mechanism topromote hyperclustering ofDR5 in a targetedmanner. The tumor-associated stroma contributes to a significant proportion of themass of many malignancies (14) and the stromal fibroblasts playan important role in the development, growth and metastasis ofcarcinomas (40–42). FAP, normally restricted in healthy adulttissue (except in granulation tissue, e.g., duringwoundhealing), isexpressed in tumor stromal fibroblasts of more than 90% ofepithelial cancers (15, 16, 43), and therefore is an ideal candidatefor targeted delivery of cancer therapeutics. The FAP-DR5 BsAbswe described here, including RG7386, exhibited high specificity

Figure 5.RG7386 demonstrates in vivo efficacy and additive effects in combination with irinotecan in a colorectal cancer (CRC) PDX model. A, IHC analysis of FAPexpression in a subcutaneous Co5896 PDX model. B, analysis of Co5896 TGI (median tumor volume and IQR; n ¼ 10 animals/group) in response toRG7386 (twice weekly administration). C, combination of RG7386 and irinotecan treatment of colorectal cancer PDX tumors. (Median tumor volumeand IQR; n ¼ 10 animals/group). Median tumor volume for vehicle-treated animals extends beyond the y-axis shown here.

Br€unker et al.





and a clear dependency on FAP-based cross-linking to promotetumor cell death. In vitro, apoptosis induction of tumor target cellsonly occurred in the presence of FAP-expressing fibroblasts andreplacement of the FAP CrossFab unit with a non-binding IgG(DP47GS) was sufficient to prevent tumor cell apoptosis. Inaddition, the FAP-dependent mode of BsAb action successfullytranslated into in vivo efficacy in several xenograft models, includ-ing a colorectal cancer PDX and a sarcoma PDX model. Theantitumor effects observed in the sarcoma PDX model alsoindicate that RG7386 function is not restricted to an environmentconsisting of a FAP-positive stroma, but can also be applied tomesenchymal-derived tumors that express FAP directly on malig-nant cells.

The broad application of our FAP-DR5 bispecific moleculeswas evident in five different tumor cell lines (lung, renal,colorectal, and breast cancer cells). However, the degree ofapoptosis was different for each cell line and did not necessarilycorrelate with DR5 expression levels, indicating that there areadditional factors influencing this mechanism. In some cellsthe DR-initiated signal requires amplification to induce max-imum apoptosis. Correspondingly, we demonstrate increasedinhibition of tumor growth in patient-derived colorectal cancerand sarcoma models in the presence of both RG7386 withirinotecan or with doxorubicin, respectively, which may beindicative of synergistic extrinsic and intrinsic apoptosis sig-

naling (7, 44). It is, however, important to note that p53, animportant tumor suppressor and a key regulator of the intrinsicpathway, is inactivated in many human cancers and the highprevalence of p53 mutants (dominant negative forms) contri-butes significantly to chemotherapeutic resistance (44–46). Asa consequence, activation of the extrinsic pathway offers anattractive target for cancer therapy as the apoptotic cascade isp53 independent, and hence not susceptible to development ofapoptosis resistance (47).

Taken together, our preclinical data strongly suggest thatRG7386, a novel FAP-DR5 tetravalent BsAb, is superior to existingDR5 agonists as it offers a targeted strategy to specifically activatethe extrinsic apoptosis pathway in tumor cells by avidity-drivenDR5 hyperclustering. Future studies will be essential for clinicaldevelopment of RG7386 particularly for the successful applica-tion of combination therapies.

Disclosure of Potential Conflicts of InterestC. Klein has ownership interest (including patents) in Roche. No potential

conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: P. Br€unker, K. Wartha, T. Friess, S. Grau-Richards,B. Weiser, H. Niu, K. Packman, C. Klein, P. Uma~na

Figure 6.RG7386 inhibits growth of FAP-positive mesenchymal tumors. A,RG7386-mediated antitumor efficacywas monitored in a FAP-positive LOX-IMVI desmoplastic melanoma cell line(median tumor volume and IQR; n¼ 6animals/group) and in a Sarc4605sarcoma PDX model (median tumorvolume and IQR; n ¼ 10 animals/group; B). C, combination of RG7386and doxorubicin treatment ofSarc4605 tumors (median tumorvolume and IQR; n ¼ 10 animals/group). Median tumor volume forvehicle-treated animals extendsbeyond the y-axis shown here.






Development of methodology: P. Br€unker, T. Friess, S. Grau-Richards,M. Majety, V. Runza, H. Niu, K. Packman, N. Feng, S. Daouti, E. M€ossner,T.G. Weber, W. Scheuer, H. Sade, C. Shao, B. Liu, P. Wang, C. Klein,P. Uma~naAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Friess, S. Grau-Richards, I. Waldhauer, C.F. Koller,M. Majety, H. Niu, N. Feng, S. Daouti, R.J. Hosse, E. M€ossner, T.G. Weber,F. Herting, H. Sade, C. Shao, B. Liu, P. Wang, G. Xu, S. Vega-Harring, K. Bosslet,P. Uma~naAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P. Br€unker, K. Wartha, T. Friess, S. Grau-Richards,I. Waldhauer, C.F. Koller, B. Weiser, M. Majety, V. Runza, H. Niu, S. Daouti,R.J. Hosse, E. M€ossner, T.G. Weber, F. Herting, W. Scheuer, H. Sade, C. Shao,G. Xu, S. Vega-Harring, P. Uma~naWriting, review, and/or revision of the manuscript: P. Br€unker, T. Friess,S. Grau-Richards, I. Waldhauer, C.F. Koller, M. Majety, V. Runza, H. Niu,F. Herting, S. Vega-Harring, C. Klein, K. Bosslet, P. Uma~na

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): P. Br€unker, S. Grau-Richards, B. Weiser,E. M€ossner, F. Herting, C. Shao, S. Vega-Harring, K. Bosslet, P. Uma~naStudy supervision: T. Friess, M.Majety, H. Niu, F. Herting, K. Bosslet, P. Uma~naOther (conducted preclinical studies and histologic analysis of explantedtumor tissue): W. Scheuer

AcknowledgmentsAll authors were employees of Roche at the time of this study. The authors

thank Aurexel Ltd. (www.aurexel.com) for editorial support funded by Roche.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 11, 2015; revised January 15, 2016; accepted January 18,2016; published OnlineFirst April 1, 2016.

References1. Ashkenazi A,Dixit VM.Death receptors: signaling andmodulation. Science

1998;281:1305–8.2. Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor super-

families: integrating mammalian biology. Cell 2001;104:487–501.3. Wilson NS, Yang A, Yang B, Couto S, Stern H, Gogineni A, et al. Proa-

poptotic activation of death receptor 5 on tumor endothelial cells disruptsthe vasculature and reduces tumor growth. Cancer Cell 2012;22:80–90.

4. Yang A, Wilson NS, Ashkenazi A. Proapoptotic DR4 and DR5 signalingin cancer cells: toward clinical translation. Curr Opin Cell Biol 2010;22:837–44.

5. Herbst RS, Kurzrock R, Hong DS, Valdivieso M, Hsu CP, Goyal L, et al. Afirst-in-human study of conatumumab in adult patients with advancedsolid tumors. Clin Cancer Res 2010;16:5883–91.

6. Kindler HL, Richards DA, Garbo LE, Garon EB, Stephenson JJ Jr., Rocha-Lima CM, et al. A randomized, placebo-controlled phase 2 study ofganitumab (AMG 479) or conatumumab (AMG 655) in combinationwith gemcitabine in patients with metastatic pancreatic cancer. Ann Oncol2012;23:2834–42.

7. Wiezorek J, Holland P, Graves J. Death receptor agonists as a targetedtherapy for cancer. Clin Cancer Res 2010;16:1701–8.

8. Adams C, Totpal K, Lawrence D, Marsters S, Pitti R, Yee S, et al. Structuraland functional analysis of the interaction between the agonistic mono-clonal antibody Apomab and the proapoptotic receptor DR5. Cell DeathDiffer 2008;15:751–61.

9. Chuntharapai A, Dodge K, Grimmer K, Schroeder K, Marsters SA,Koeppen H, et al. Isotype-dependent inhibition of tumor growth invivo by monoclonal antibodies to death receptor 4. J Immunol 2001;166:4891–8.

10. Motoki K, Mori E, Matsumoto A, Thomas M, Tomura T, Hum-phreys R, et al. Enhanced apoptosis and tumor regression inducedby a direct agonist antibody to tumor necrosis factor-relatedapoptosis-inducing ligand receptor 2. Clin Cancer Res 2005;11:3126–35.

11. Takeda K, Yamaguchi N, Akiba H, Kojima Y, Hayakawa Y, Tanner JE, et al.Induction of tumor-specific T-cell immunity by anti-DR5 antibody ther-apy. J Exp Med 2004;199:437–48.

12. Kim JM, Ashkenazi A. Fcgamma receptors enable anticancer action ofproapoptotic and immune-modulatory antibodies. J Exp Med 2013;210:1647–51.

13. Wilson NS, Yang B, Yang A, Loeser S, Marsters S, Lawrence D, et al. AnFcgamma receptor-dependent mechanism drives antibody-mediated tar-get-receptor signaling in cancer cells. Cancer Cell 2011;19:101–13.

14. Brennen WN, Isaacs JT, Denmeade SR. Rationale behind targeting fibro-blast activation protein-expressing carcinoma-associated fibroblasts as anovel chemotherapeutic strategy. Mol Cancer Ther 2012;11:257–66.

15. Garin-Chesa P, Old LJ, Rettig WJ. Cell surface glycoprotein of reactivestromal fibroblasts as a potential antibody target in human epithelialcancers. Proc Natl Acad Sci U S A 1990;87:7235–9.

16. Rettig WJ, Garin-Chesa P, Healey JH, Su SL, Ozer HL, Schwab M, et al.Regulation and heteromeric structure of the fibroblast activation protein in

normal and transformed cells of mesenchymal and neuroectodermalorigin. Cancer Res 1993;53:3327–35.

17. Schaefer W, Regula JT, Bahner M, Schanzer J, Croasdale R, Durr H, et al.Immunoglobulin domain crossover as a generic approach for the produc-tion of bispecific IgG antibodies. Proc Natl Acad Sci U S A 2011;108:11187–92.

18. Hessell AJ, Hangartner L, Hunter M, Havenith CE, Beurskens FJ, Bakker JM,et al. Fc receptor but not complement binding is important in antibodyprotection against HIV. Nature 2007;449:101–4.

19. Weber TG, Osl F, Renner A, Poschinger T, Galban S, Rehemtulla A, et al.Apoptosis imaging for monitoring DR5 antibody accumulation andpharmacodynamics in brain tumors noninvasively. Cancer Res 2014;74:1913–23.

20. Weber TG, Poschinger T, Galban S, Rehemtulla A, Scheuer W. Noninvasivemonitoring of pharmacodynamics and kinetics of a death receptor 5antibody and its enhanced apoptosis induction in sequential applicationwith doxorubicin. Neoplasia 2013;15:863–74.

21. Ridgway JB, Presta LG, Carter P. `Knobs-into-holes' engineering of anti-bodyCH3domains for heavy chain heterodimerization. Protein Eng 1996;9:617–21.

22. Marconi M, Ascione B, Ciarlo L, Vona R, Garofalo T, Sorice M, et al.Constitutive localization of DR4 in lipid rafts is mandatory for TRAIL-induced apoptosis in B-cell hematologic malignancies. Cell Death Dis2013;4:e863.

23. Xu Y, Szalai AJ, Zhou T, Zinn KR, Chaudhuri TR, Li X, et al. Fc gamma Rsmodulate cytotoxicity of anti-Fas antibodies: implications for agonisticantibody-based therapeutics. J Immunol 2003;171:562–8.

24. Li F, Ravetch JV. Apoptotic and antitumor activity of death receptorantibodies require inhibitory Fcgamma receptor engagement. Proc NatlAcad Sci U S A 2012;109:10966–71.

25. Kaplan-Lefko PJ, Graves JD, Zoog SJ, Pan Y, Wall J, Branstetter DG, et al.Conatumumab, a fully human agonist antibody to death receptor 5,induces apoptosis via caspase activation in multiple tumor types. CancerBiol Ther 2010;9:618–31.

26. Doi T,MurakamiH,Ohtsu A, FuseN, Yoshino T, YamamotoN, et al. Phase1 study of conatumumab, a pro-apoptotic death receptor 5 agonist anti-body, in Japanese patients with advanced solid tumors. Cancer ChemotherPharmacol 2011;68:733–41.

27. Plummer R, Attard G, Pacey S, Li L, Razak A, Perrett R, et al. Phase 1 andpharmacokinetic study of lexatumumab in patientswith advanced cancers.Clin Cancer Res 2007;13:6187–94.

28. Wakelee HA, Patnaik A, Sikic BI, Mita M, Fox NL, Miceli R, et al. Phase Iand pharmacokinetic study of lexatumumab (HGS-ETR2) given every 2weeks in patients with advanced solid tumors. Ann Oncol 2010;21:376–81.

29. Fay F, McLaughlin KM, Small DM, Fennell DA, Johnston PG, Longley DB,et al. Conatumumab (AMG 655) coated nanoparticles for targeted pro-apoptotic drug delivery. Biomaterials 2011;32:8645–53.

30. Gieffers C, KlugeM,Merz C, Sykora J, ThiemannM, Schaal R, et al. APG350induces superior clustering of TRAIL receptors and shows therapeutic


Br€unker et al.




antitumor efficacy independent of cross-linking via Fcgamma receptors.Mol Cancer Ther 2013;12:2735–47.

31. Allen JE, Ferrini R, Dicker DT, Batzer G, Chen E, Oltean DI, et al. TargetingTRAIL death receptor 4 with trivalent DR4 Atrimer complexes. Mol CancerTher 2012;11:2087–95.

32. Huet HA, Growney JD, Johnson JA, Li J, Bilic S, Ostrom L, et al.Multivalent nanobodies targeting death receptor 5 elicit superiortumor cell killing through efficient caspase induction. MAbs 2014;6:1560–70.

33. Swers JS, Grinberg L, Wang L, Feng H, Lekstrom K, Carrasco R, et al.Multivalent scaffold proteins as superagonists of TRAIL receptor 2-inducedapoptosis. Mol Cancer Ther 2013;12:1235–44.

34. Papadopoulos KP, Isaacs R, Bilic S, Kentsch K, Huet HA, HofmannM, et al.Unexpected hepatotoxicity in a phase I study of TAS266, a novel tetravalentagonistic Nanobody(R) targeting the DR5 receptor. Cancer ChemotherPharmacol 2015;75:887–95.

35. de Bruyn M, Rybczynska AA, Wei Y, Schwenkert M, Fey GH, Dierckx RA,et al. Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP)-targeted delivery of soluble TRAIL potently inhibits melanoma outgrowthin vitro and in vivo. Mol Cancer 2010;9:301–14.

36. Lawrence D, Shahrokh Z, Marsters S, Achilles K, Shih D, Mounho B, et al.Differential hepatocyte toxicity of recombinant Apo2L/TRAIL versions. NatMed 2001;7:383–5.

37. Siegemund M, Pollak N, Seifert O, Wahl K, Hanak K, Vogel A, et al.Superior antitumoral activity of dimerized targeted single-chain TRAILfusion proteins under retention of tumor selectivity. Cell Death Dis2012;3:e295.

38. Spitzer D,McDunn JE, Plambeck-Suess S, Goedegebuure PS, Hotchkiss RS,Hawkins WG. A genetically encoded multifunctional TRAIL trimer facil-itates cell-specific targeting and tumor cell killing. Mol Cancer Ther 2010;9:2142–51.

39. StuckeyDW, Shah K. TRAIL on trial: preclinical advances in cancer therapy.Trends Mol Med 2013;19:685–94.

40. Hewitt RE, Powe DG, Carter GI, Turner DR. Desmoplasia and its relevanceto colorectal tumour invasion. Int J Cancer 1993;53:62–9.

41. Pandol S, Edderkaoui M, Gukovsky I, Lugea A, Gukovskaya A. Desmo-plasia of pancreatic ductal adenocarcinoma. Clin Gastroenterol Hepatol2009;7:S44–7.

42. Ronnov-Jessen L, Petersen OW, Bissell MJ. Cellular changes involved inconversion of normal to malignant breast: importance of the stromalreaction. Physiol Rev 1996;76:69–125.

43. Tuxhorn JA, Ayala GE, SmithMJ, Smith VC, Dang TD, Rowley DR. Reactivestroma in human prostate cancer: induction of myofibroblast phenotypeand extracellular matrix remodeling. Clin Cancer Res 2002;8:2912–23.

44. Lee JM, Bernstein A. Apoptosis, cancer, and the p53 tumour suppressorgene. Cancer Metastasis Rev 1995;14:149–61.

45. Hollstein M, Rice K, Greenblatt MS, Soussi T, Fuchs R, Sorlie T, et al.Database of p53 gene somatic mutations in human tumors and cell lines.Nucleic Acids Res 1994;22:3551–5.

46. Igney FH, Krammer PH. Death and anti-death: tumour resistance toapoptosis. Nat Rev Cancer 2002;2:277–88.

47. Galligan L, Longley DB, McEwan M, Wilson TR, McLaughlin K, JohnstonPG. Chemotherapy and TRAIL-mediated colon cancer cell death: the rolesof p53, TRAIL receptors, and c-FLIP. Mol Cancer Ther 2005;4:2026–36.






2016;15:946-957. Published OnlineFirst April 1, 2016.Mol Cancer Ther Peter Brünker, Katharina Wartha, Thomas Friess, et al. Tumor Cell ApoptosisTriggers FAP-Dependent, Avidity-Driven DR5 Hyperclustering and RG7386, a Novel Tetravalent FAP-DR5 Antibody, Effectively

Updated version

10.1158/1535-7163.MCT-15-0647doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2016/04/01/1535-7163.MCT-15-0647.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/15/5/946.full#ref-list-1

This article cites 47 articles, 21 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/15/5/946To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-15-0647

http://mct.aacrjournals.org/content/suppl/2016/04/01/1535-7163.MCT-15-0647.DC1

http://mct.aacrjournals.org/content/15/5/946.full#ref-list-1

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/15/5/946


rg7386, a novel tetravalent fap-dr5 antibody,...

Documents