antitumor efﬁcacy of a bispeciﬁc antibody that targets...

Therapeutics, Targets, and Chemical Biology

Antitumor Efficacy of a Bispecific Antibody That TargetsHER2 and Activates T Cells

Teemu T. Junttila, Ji Li, Jennifer Johnston, Maria Hristopoulos, Robyn Clark, Diego Ellerman, Bu-Er Wang,Yijin Li, Mary Mathieu, Guangmin Li, Judy Young, Elizabeth Luis, Gail Lewis Phillips, Eric Stefanich,Christoph Spiess, Andrew Polson, Bryan Irving, Justin M. Scheer, Melissa R. Junttila, Mark S. Dennis,Robert Kelley, Klara Totpal, and Allen Ebens

AbstractClinical results from the latest strategies for T-cell activation in cancer have fired interest in combination

immunotherapies that can fully engage T-cell immunity. In this study, we describe a trastuzumab-based bispecificantibody, HER2-TDB, which targets HER2 and conditionally activates T cells. HER2-TDB specifically killed HER2-expressing cancer cells at low picomolar concentrations. Because of its unique mechanism of action, which isindependent of HER2 signaling or chemotherapeutic sensitivity, HER2-TDB eliminated cells refractory tocurrently approved HER2 therapies. HER2-TDB exhibited potent antitumor activity in four preclinical modelsystems, including MMTV-huHER2 and huCD3 transgenic mice. PD-L1 expression in tumors limited HER2-TDBactivity, but this resistance could be reversed by anti–PD-L1 treatment. Thus, combining HER2-TDB with anti–PD-L1 yielded a combination immunotherapy that enhanced tumor growth inhibition, increasing the rates anddurability of therapeutic response. Cancer Res; 74(19); 5561–71. �2014 AACR.

IntroductionThe recent approval of ipilimumab (1) and exciting

responses observed during clinical trials of PD-1 and PD-L1antibodies (2) clearly illustrate the potential of T cell–targetingcancer immunotherapies. The success of strategies that rein-vigorate T-cell activity depends on modulation of multiplestimulatory and inhibitory events that enable an antitumorimmune response (2). An attractive alternative of leveraging T-cell activity to eliminate cancer is to not rely on existing tumorimmune response, but rather to induce T cells to kill tumorcells directly by generating new tumor specificities. Bispecificantibodies can be used to broadly harness the antitumorcapacity of T-cell immunity (3). However, successful clinicaluse of modified and reengineered antibodies and antibodyfragments is far from trivial (4). To progress a drug candidatefrom laboratory to clinic, a vast array of requirements needs tobemet in regard to "drug-like" properties. Immunogenicity andshort serum half-life are additional problems for bispecificmolecules with modified antibody sequences and antibodyfragment–based platforms.Although several tumor targets and bispecific antibody

platforms have demonstrated generalflexibility and preclinicalfeasibility for this approach, very few molecules have been

implemented in clinical use. In 2009, the European MedicinesAgency (EMA) approved the use of a trifunctional EpCam �CD3 bispecific antibody, catumaxomab (Removab), for theintraperitoneal treatment of malignant ascites (5). Promisingclinical activity has also been demonstrated with a CD19 �CD3–targeting bispecific scFv antibody fragment, blinatumo-mab (3). Both catumaxomab and blinatumomab illustrate howtechnological challenges affect clinical use of bispecific anti-bodies. Catumaxomab is a mouse IgG2a/rat IgG2b hybrid, andthus it is highly immunogenic in human. Dosing of catumax-omab is limited to maximum of four doses and 20 days.Because of the extremely short serum half-life (1.25 hours) ofblinatumomab (3), it is continuously infused via pumpthroughout the treatment cycle.

Here, we report the properties of a T cell–dependent bis-pecific antibody targeting HER2 (HER2-TDB), which caninduce a polyclonal T-cell response to tumors. The responsedoes not require a preexisting tumor immune response and ithas reduced potential for immune escape (e.g., loss of MHCexpression). Themodality combines extreme potency of the T-cell activity with favorable drug-like properties of IgG1 mol-ecule, including long half-life.

Despite recent advances in treatment of HER2þ breastcancer (6–9), several resistance mechanisms have been iden-tified that engage redundant survival signaling pathways (10–12). De novo or acquired resistance is an expected outcomealso for a subset of T-DM1 (ado-trastuzumab emtansine,Kadcyla)–treated patients. Therefore, a significant unmetmedical need remains for HER2þ breast cancer. HER2-TDBkills all tested HER2þ tumor cells with low picomolar EC50,and due to its different mechanism of action, effectively killscells that are refractory to trastuzumab, lapatinib, and T-DM1.

Genentech, Inc., South San Francisco, California.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Teemu T. Junttila, Genentech, Inc., 1 DNA Way,Mailstop 231a, South San Francisco, CA 94080. Phone: 650-225-1000;Fax: 650-225-5214; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-3622-T

�2014 American Association for Cancer Research.

CancerResearch

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An important outstanding question with T cell–engagingbispecific antibodies is whether, they toomay be susceptible toT cell–suppressive resistance mechanisms following the initialT-cell response? Our results demonstrate that PD-1/PD-L1 caninhibit T-cell killing activity induced by bispecific antibodies.Our results using immunocompetent huCD3-transgenic ani-mal model further suggest that combining T cell–recruitingantibodies with anti–PD-L1 antibodies improves outcome ofthe treatment.

Materials and MethodsAntibody expression and purification

The "knob" arm of HER2 huIgG1 TDB is humanized anti-HER2 4D5 (trastuzumab; ref. 13) and "hole" arm is humanizedanti-CD3 UCHT1.v9 (14, 15). The huIgG1 bispecific antibodieswere produced by two different approaches as describedearlier (16): coculture of bacteria expressing each of the twoantibody arms, or by expressing each arm separately and thenannealing them in vitro.

To avoid immune response toward the TDB, amurine IgG2a(knob-hole, D265A, and N297G) isotype HER2-TDB expressedin Chinese hamster ovary (CHO) cells was used in experimentswith immunocompetent mice. In muIgG2a HER2-TDBs, the"knob" arm is murine anti-HER2 4D5 and the "hole" is eitherchimeric anti-murine CD3 2C11 (4D5/2C11-TDB; ref. 17) ormouse anti-hu CD3 SP34 (4D5/SP34-TDB; ref. 18). Bispecificantibody purification is described elsewhere (16).

Antibody characterizationThe molecular weight of the bispecific antibody was ana-

lyzed by mass spectrometry [liquid chromatography-electro-spray ionization/time of flight (LC-ESI/TOF)] as describedbefore (19). The antibodies were also analyzed by analytic sizeexclusion chromatography in a Zenix SEC-300 column (SepaxTechnologies) using an Agilent 1100 HPLC system (AgilentTechnologies). The presence of residual antibody fragmentswas quantified by electrophoresis using a 2100 Bioanalyzer anda Protein 230 Chip (Agilent Technologies).

HER2-TDB affinityThe competitive Scatchard assay is described in detail

elsewhere (20).

Breast cancer cell proliferationBreast cancer cell proliferation/viability was detected

using the CellTiter-Glo Luminescent Cell Viability Assay(Promega). For the assay, 5 � 103 cells per well were platedin 96-well plates and incubated overnight for cell attachmentbefore treatments.

Blood cell fractionationPeripheral bloodmononuclear cells (PBMC) were separated

from the blood of healthy volunteers using lymphocyte sepa-ration medium (MP Biomedicals). CD8þ cells were extractedfrom PBMC using human CD8þ Isolation Kit from MiltenyiBiotec (#130-094-156) by negative selection. CD3� depletionwas done using CD3þ MicroBeads fromMiltenyi Biotec (#130-050-101).

In vitro cytotoxicity assays (in vitro ADCC, T-cell killing)In vitro cytotoxicity assays [Cytotoxicity Detection Kit; lac-

tate dehydrogenase (LDH); Roche] were performed as previ-ously described (21). Alternatively, in vitro cytotoxicity wasmonitored by flow cytometry. Target cells were labeled withCFSE (Invitrogen; #C34554). The labeled target cells and CD8þ

cells were mixed with or without TDB for 4 to 26 hours. At theend of the incubation, the cells were lifted by trypsin andcollected from the plate. The cells were resuspended in equalvolume of PBS þ 2% FBS þ 1 mmol/L EDTA þ propidiumiodine (PI). Flow cytometry analysis was done on a FACSCa-libur in automation format. The number of live target cells wascounted by gating on CFSEþ/PI� cells. The percentage ofcytotoxicity was calculated as follows: %cytotoxicity (livetarget cell number without TDB � live target cell numberwith TDB)/(live target cell number without TDB) � 100. iCellcardiomyocytes (Cellular Dynamics International) wererevived from liquid nitrogen and plated at 27,000 cells per96-well 7 days before the assay and treated as per the man-ufacturer's instructions. CD8 cells were added in 3:1 ratio oniCells for 24 hours together with the treatment. After 24 hours,cells were gently washed twice with PBS to remove T cells andviability was measured using the CellTiter-Glo Assay.

Analysis of T-cell activationCells were stained with CD8-FITC (BD Biosciences; 555634),

CD69-PE (BD Biosciences; 555531), and CD107a-Alexa Fluor647 (eBioscience; 51-1079). Alternatively, cells were fixedand permeabilized with Cytofix/CytoPerm solution (BD Bio-sciences; 554722) and stained with anti-granzyme B-AlexaFluor 647 (BD Biosciences; 560212).

Detection of soluble granzymes and perforinSoluble perforin (Cell Sciences), granzyme A, and granzyme

B (eBioscience) were detected from growth media by ELISAaccording to the manufacturer's protocols.

PD-1 induction and effect of PD-L1 expression on TDBactivity

Purified CD8þ T cells from human peripheral blood wereprimed with 100 mg/mL of HER2-TDB and SKBR3 cell at 3:1ratio for 24 hours. After 24 hours of incubation, the cell pelletwas digested with Non-Enzyme Cell Dissociation Solution(Sigma; #C5789) at 37�C for 10 minutes and CD8þ T cells wererecovered using Human CD8þ MicroBeads (Miltenyi Biotec;#130-045-201). The primed-CD8þ T cells were used for in vitrocytotoxicity assay. Inflat-bottomed 96-well plate, CFSE-labeled293 cells or 293-PD-L1 cells were mixed with primed effectorcells in 3:1 ratio in the presence or absence of HER2-TDB and10 mg/mL anti–PD-L1 antibody (clone 6E11, mIgG2A, D265A,and N297A; Genentech). After 24 hours, cytotoxicity was mea-sured by counting live CFSEþ target cells by flow cytometry.

Pharmacokinetic study in ratsSprague-Dawley rats (n¼ 4/group) were dosed intravenous

(i.v.) bolus either HER2-TDB or trastuzumab at 10 mg/kg.Serum samples were assayed for human IgG by ELISA andpharmacokinetic (PK) parameters were determined with a

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two-compartmentmethod (Model 7) usingWinNonlin, version5.2.1 (Pharsight Corp.).

In vivo efficacyDosing and monitoring were performed in accordance

with guidelines from the Institutional Animal Care and UseCommittee at Genentech, Inc. NOD/SCID mice (NOD.CB17-Prkdcscid/J; The Jackson Laboratory West) were implantedwith 0.36 mg, 60-day sustained release estrogen pellets (Inno-vative Research of America) 1 to 3 days before cell inoculation,subcutaneously over the opposite flank of tumor inoculation.On day 0, 5 million MCF7 neo/HER2 and 10 million nonacti-vated human PBMCs (huPBMC) in HBSS-Matrigel were inoc-ulated in the right second/third mammary fat pad. The firsttreatments were administered 2 hours after inoculation. Alltreatments were administered 1�/week by i.v. tail vein injec-tion for a total of three doses. MMTV.huHER2 transgenicanimals maintained on a FVB/N background have been pre-viously described (22). For experiments with syngeneic tumors,0.1 million CT26-HER2 cells were injected subcutaneously toBalb/c or human CD3e transgenic mice (23). To avoid immuneresponse toward theTDB, amurine IgG2A version of theHER2-TDB was used in experiments with immunocompetent mice.Anti–PD-L1 antibody clone 25A1 (mIgG2A, D265A, and N297A;Genentech) was used for therapeutic blockade of PD-L1.

ResultsGeneration and purification of full-length HER2-CD3bispecific antibody (HER2-TDB) using knobs-into-holestechnologyHER2-TDB was generated using a knobs-into-holes strategy

(Fig. 1A; ref. 14). The anti-CD3 arm (UCHT1.v9; hole) and theanti-HER2 arm (4D5; trastuzumab; knob) were expressed inseparate Escherichia coli cultures and later combined, or alter-natively, cocultured from the start (Fig. 1B). The fully assembledantibody was isolated on Protein A and then purified fromantibody fragments by hydrophobic interaction chromatogra-phy. Size exclusion chromatography showed a very low level ofaggregation (Fig. 1C, <0.2%–0.9%) andmass spectrometry anal-ysis showed a main mass deconvolution peak corresponding tothe heterodimer with an absence of significant amounts ofeither homodimer (Fig. 1D). These results demonstrate thathigh-quality HER2-TDB can be efficiently produced using stan-dard expression and purification methods.

T cell–independent properties of HER2-TDBTarget arm binding affinity of HER2-TDB by Scatchard

analysis (KD ¼ 5.4 nmol/L; Fig. 1E) was similar to monovalenttrastuzumabFab(KD¼ 3.9nmol/L) and lower than theaffinityofbivalent trastuzumab toHER2(KD¼ 0.7nmol/L).TheKD forCD3arm binding affinity to Jurkat cells was 4.7 nmol/L (not shown).The ability of HER2-TDB to directly inhibit SKBR3 proliferationwas reduced as compared with bivalent trastuzumab (Fig. 1F).Antibodies produced in E. coli are not glycosylated, whichresults in impaired FcgR binding, which is required to mediateantibody-dependent cell-mediated cytotoxicity (ADCC; refs. 24,25).E. coliproduced trastuzumabandHER2-TDBwere unable toinduce natural killer cell–mediated ADCC (Fig. 1G).

Target-dependent T-cell activation and cytotoxicityT-cell activation was not detected when CD8þ cells were

incubated with HER2-TDB or target cells that do not expresshuman HER2 (BJAB cells; Fig. 2A). A robust T-cell activationwas seen when HER2þ SKBR3 cells were used as targetsaccompanied by release of cytotoxic granules. Soluble perforin,granzyme A, and granzyme B were detected in the growthmedia by ELISA (Fig. 2B), but onlywhen all the key components(HER2-TDB, T cells, and HER2-expressing cells) were includedin the reaction. Granule exocytosis coincided with significantHER2-TDB–induced elevation of caspase-3/7 activity, apopto-sis, and cytotoxicity (Fig. 2C).

HER2-TDB does not mediate killing of vector-transfected3T3-cells (Fig. 2D), in contrast, the HER2-transfected 3T3-cellswere very efficiently killed. Addition of HER2-ECD or trastu-zumab Fab to the killing assay efficiently inhibited the killingactivity (Fig. 2E). To confirm T-cell dependence of killing, wedepleted CD3þ cells from the PBMC (Fig. 2F). The depletionresulted in loss of target cell killing activity.

Early signs of T-cell activation (CD69) were detectable 4hours after HER2-TDB treatment was initiated (Supplemen-tary Fig. S1A). However, late activation markers (extracellularCD107a) were not significantly expressed until the 24-hourtime point. Activation of T cells was reflected in killing ofHER2þ breast cancer cells (Supplementary Fig. S1A).

These results show that activation of T cells by HER2-TDB isconditional and target dependent. Binding of HER2-TDB to Tcells in the absence of target cells is not sufficient to activate Tcells.

HER2-TDB induces T-cell proliferationCytotoxicity was significantly reduced by effector cell titra-

tion (Supplementary Fig. S1B). However, even with an E:T ratioof�1:1, a weak LDH signal and robust activation of T cells wasdetected. We next investigated whether HER2-TDB induces T-cell proliferation by coculturing CD8þ T cells, target cells(SKBR3), and 0.1 mg/mL HER2-TDB followed by T-cell culturein the absence of target cells and HER2-TDB. After 3 days, 75%of the T cells pulsed with TDB and target cells had undergone acell division (Supplementary Fig. S2); however, the cell numberdid not increase. Supplementing the growthmediawith IL2 (20ng/mL) provided a survival signal to CD8þ cells, and a robustaccumulation of T cells was detected, but only if they wereexposed to both HER2-TDB and target cells (SupplementaryFig. S2).

HER2-TDB activity correlates with the target cell HER2expression level

To investigate the relationship between target copy numberand TDB activity, we selected a panel of cancer cell lines withpredetermined number of HER2 receptors on the cell mem-brane (Fig. 3A and G; ref. 26). HER2-amplified/overexpressingcell lines were significantly more sensitive to the TDB-medi-ated killing (P ¼ 0.007, t test) and were efficiently lysed atfemtomolar to low picomolar concentrations (EC50 ¼ 0.8–3pmol/L; Fig. 3B). Cell lines expressing low levels of HER2 weresignificantly less sensitive to HER2-TDB antibody (EC50 ¼ 33–51 pmol/L). Fewer than 1,000 copies of target antigen were

Full-Length HER2-TDB

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sufficient to support T-cell killing.We have previously reportedEC50 4,500 pmol/L as the sensitivity of SKBR-3 to trastuzumabin a 5-day viability assay.

Several normal human tissues express low levels of HER2.Expression level of HER2 in iCell cardiomyocytes (27, 28) iscomparable with MCF-7 cells and low level of in vitro activitywas detected for HER2-TDB (Fig. 3C and D).

Next, we cotargeted MCF7 (low HER2 expression), or BJABcells (noHER2 expression) withHER2-amplified SKBR3 cells inthe same killing assay. No killing of MCF7 cells was detectableat the EC50 for SKBR3 killing (Fig. 3E). No significant killing of

BJAB cellswas detectable at anyHER2-TDB concentration (Fig.3F). These results suggest that an overexpression-based ther-apeutic index may exist for HER2-TDB and demonstrate a lackof bystander killing.

Very low target occupancy is sufficient for TDB activityHER2occupancy at EC50 forHER2-TDBwas calculated using

formula [D]/[D]þKD (where the D ¼ drug and KD for HER2-TDBwas 5.4 nmol/L). In all tested cell lines, less than 1% targetoccupancy was sufficient for efficient killing (Fig. 3G), and inthe case of the high HER2-expressing cell lines, the required

Figure 1. Generation and T cell–independent properties of HER2-TDB. A, amino acid substitutions are generated to CH3 domains of the "knob" (a-HER2 4D5)and "hole" (a-CD3 UCHT1.v9) heavy chains, which selectively promote heterodimerization to generate bispecific full-length IgG1. B, overview of theTDBpurification. ProA,Protein Aaffinity purification;HIC, hydrophobic interactionchromatography;QC,quality control; SEC, sizeexclusionchromatography.C, size exclusion chromatography demonstrates low levels of aggregate or single arms. D, MS analysis indicates undetectable levels of homodimericspecies. E, binding to SKBR-3 was determined by competition binding of 125I-trastuzumab Fab with nonlabeled trastuzumab (black), trastuzumab-Fab(blue), or bispecificHER2-TDB (red). F, direct effect on proliferation of SKBR-3 cells was analyzed after 6 days of treatment with antibodies using theCellTiter-Glo Luminescent Cell Viability Assay. G, the ability of trastuzumab, trastuzumab produced in E. coli, and HER2-TDB tomediate in vitro ADCC by natural killercells was measured using assay detecting LDH released from lysed cells. Time point, 4 hours. Data points in the figure represent the mean of threesamples; error bar, SD.

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occupancy was even lower (0.01%–0.05%). The calculatedabsolute number of TDB bound to HER2 at the EC50 was aslow as 10 to 150 molecules in the low expressing cell lines.These results showcase the extreme potency of HER2-TDB andare consistent with studies of T-cell receptor triggering, whichsuggest that as few as 1 to 25 T-cell receptors need to beengaged to trigger T-cell responses (29–31).

HER2-TDB is efficient in killing of HER2þ cancer cellsrefractory to anti-HER2 therapiesWe next selected cell lines previously shown to express

high levels of HER2 and be insensitive to the direct cellulareffects of trastuzumab and lapatinib in vitro (10, 32). Sen-sitivity to T-DM1 has been previously reported (32, 33).EC50 for HER2-TDB–mediated killing was in the femtomo-lar or low picomolar range (Fig. 4A). In addition, HER2-TDBis effective in killing HER2þ lung cancer cells, demonstrat-ing that the effect of the molecule is not limited to breastcancer cells. Using two independent cell line models

(BT474; Fig. 4B and C; KPL-4, not shown), we demonstratethat acquired resistance to T-DM1 did not affect thesensitivity to HER2-TDB. These results demonstrate thatHER2-TDB displays broad subpicomolar killing activityagainst HER2þ cells regardless of the tissue of origin, PI3Kpathway activation status or sensitivity to trastuzumab,lapatinib, and T-DM1.

Pharmacokinetics of HER2-TDB in ratSprague-Dawley rats were administered a single i.v. dose of

10mg/kg of eitherHER2-TDBor trastuzumab. HER2-TDBdoesnot cross-react with rat CD3 or rat HER2 and displayed abiphasic disposition typical of an IgG1with a short distributionphase and slow elimination phase (Fig. 5). The clearance andhalf-life of HER2-TDBwerewithin expected ranges for a typicalnonbinding human IgG1 in rat and not significantly differentcompared with trastuzumab. These results demonstrate thatthe molecule has the slow clearance and the expected longin vivo half-life of a typical IgG1 antibody.

Figure 2. Target-dependent T cell–mediated cytotoxicity of HER2-TDB. A, T-cell activation wasdetected by staining cells for CD8/CD69/granzyme B followed byFACS analysis. Effectors CD8þ Tcells, target SKBR-3, E:T ratio 3:1,time point of 48 hours. Datapresented as mean of two repeats.B, soluble granzymes and perforinwere detected from the mediausing ELISA and cytotoxicity usingLDH release assay. EffectorsPBMC, target SKBR-3, E:T ratio30:1, time point of 18 hours, Abs 10ng/mL. C, elevated caspaseactivity (Caspase-3/7 Glo Assay)and apoptosis (Cell DeathDetection ELISAplus assay)correspond with LDH release aftertreatment with 1 ng/mL bispecificantibody. Effectors PBMC, targetsSKBR-3, E:T ratio 10:1, time pointof 24 hours. Error bar, SD in C–F.D, cytotoxicity on HER2 (red) orvector-transfected (blue) 3T3.Effectors PBMC, E:T ratio 10:1,time point of 19 hours LDH releaseassay. E, HER2-arm binding wasblocked using trastuzumab Fab(1 mg/mL, black) or soluble HER2extracellular domain (1 mg/mL,blue). EffectorsCD8þT cells, targetBT474, E:T ratio 5:1, time point of24 hours, LDH release assay. F,killing activity of PBMC before andafter depletion of CD3þ cells.Target SKBR3, E:T ratio 20:1, timepoint of 19 hours, FACS assay.Data points in the figure representthe mean of three samples; errorbar, SD.






HER2-TDB inhibits tumor growth in vivo inimmunocompromised mice

In vivo efficacy of HER2-TDB was tested in NOD/SCID mice.MCF7-neo/HER2 cellswere grafted togetherwith nonactivatedhuPBMCs from healthy donors to mammary fat pads of mice.Mice were dosed intravenously on a weekly schedule with 0.5mg/kg of HER2-TDB or control-TDB, starting on the day oftumor cell inoculation. HER2-TDB prevented growth of HER2-expressing tumors (Fig. 6A). As expected, no efficacy wasdetected inmice when huPBMCwere omitted (SupplementaryFig. S3A). A control TDB that shares the same CD3 arm asHER2-TDB, but has an irrelevant target arm had no effect onthe tumor growth (Supplementary Fig. S3B).

HER2-TDB causes regression of mammary tumors inhuHER2 transgenic mice

We next generated a surrogate TDB using a mouse CD3-reactive antibody clone 2C11 (17) to treat MMTV-huHER2transgenic mice (22). The in vitro activity of 4D5/2C11-TDBwas similar to human CD3-reactive HER2-TDB (Supplemen-tary Fig. S4). With exception of one mouse, 4D5/2C11-TDBresulted in tumor regression in all treated mice (Fig. 6B andC). More than 80% tumor regression was detected in 43% ofthe mice. Remarkably, responses were noted in mice withtumors that were more than 1,000 mm3 at the start of the

treatment (Fig. 6D). Tumor growth was not affected bycontrol TDBs (Fig. 6E) or by treatment with 30 mg/kgbivalent murine 4D5 (precursor of humanized 4D5; trastu-zumab; Supplementary Fig. S5).

HER2-TDB has transient antitumor activity in treatmentof syngeneic huHER2-expressing tumors

T cells from CD3e transgenic mice (CD3-TG; ref. 23) expressboth mouse and human CD3 on approximately 50% level ofrespective Balb/c mouse or human T cells (Supplementary Fig.S6). CD3-TG T cells can kill human HER2-expressing targetcells in vitro (Supplementary Fig. S4), although killing activity ofmouse splenic T cells is consistently lower compared withhuman peripheral T cells. Human HER2-transfected CT26tumor cells were grown in the CD3-TG mice subcutaneouslyand established tumors were treated with weekly 0.5 mg/kg i.v.doses of HER2-TDB. HER2-TDB clearly inhibited the growth ofestablished tumors, but the effect was transient (Fig. 6F). Theactivity of HER2-TDB is dependent on T cells, because HER2-TDBhadno effect in non-CD3 transgenicmice (SupplementaryFig. S6). The in vivo responses detected in Balb/c mice using4D5/2C11-TDB were similar to the responses seen in CD3-TGmicewith human-specificCD3 arm–basedTDB (Fig. 6F andG).HER2-TDB significantly prolonged the time to tumor progres-sion (log-rank, P < 0.0001).

Figure 3. HER2-TDB activitycorrelates with the target cell HER2expression level. A, HER2 levels incancer cells were detected byWestern blot analysis. B,cytotoxicity was detected usingLDH release assay. EffectorsPBMCs, E:T ratio 25:1, time pointof 26 hours. C, HER2 levels in celllines were detected by FACS. D, invitro activity of HER2-TDB on iCellcardiomyocytes. Effectors CD8þ Tcells, E:T ratio 3:1, time point of 24hours. MCF-7 (E) or BJAB (F) cellswere labeled with CFSE and mixedwith SKBR3 and PBMC (E:T, 20:1)followed by 19-hour treatment withHER2-TDB.Cellswere stainedwithanti-HER2 allophycocyanin (APC)andPI. Thenumber of livingSKBR3(HER2 high, PI�) and MCF7 (E) orBJAB (F; CFSEþ, PI�) cells wereanalyzed by FACS and normalizedto fluorescent beads. G, HER2copy numbers were previouslyreported (26). EC50 values werecalculated from dose responsedata in Fig. 4B.Calculation ofHER2occupancy is described in text.Data points in the figure representthe mean of three samples; errorbar, SD.

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PD-L1 expression in target cells inhibits HER2-TDBactivityWe analyzed the cellular composition of the CT26-HER2

tumors to search for an explanation for incomplete tumorresponse. Of note, 10% to 30% of CD45þ cells in CT26-HER2tumors were CD8þ T cells (Supplementary Figs. S7 and S8).Almost all T cells displayed markers of activation and werepositive for PD-1 (80%–95% CD69þ, 95% PD-1þ). All CD45�

cells were positive for PD-L1, suggesting that PD-L1 expres-sion by the tumor cells may potentially inhibit the activity ofthe HER2-TDB. We functionally tested this using human Tcells. A clear upregulation of PD-1 in T cells was detected

upon overnight coculture with SKBR3 cells and HER2-TDB(Fig. 7A). Induced T cells expressing PD-1 were then trans-ferred on PD-L1 or vector-transfected 293 cells. Because 293cells express low levels of HER2, the primed T cells efficientlykilled the 293 cells, but only when the HER2-TDB was added(Fig. 7B). Expression of PD-L1 in 293 cells significantlyinhibited the killing activity and this inhibition wascompletely reversed by PD-L1–blocking antibody. Togetherthese results demonstrate that PD-L1 expression by thetarget cells can inhibit HER2-TDB activity and provide arationale for therapeutic HER2-TDB and anti–PD-L1combination.

Figure 5. PK profile of HER2-TDB.Single i.v. doses of 10 mg/kgtrastuzumab (open symbols) orHER2-TDB (black symbols) wereinjected into Sprague-Dawleyrats. Serum samples were assayedfor test agent by ELISA. Mean �SD. HER2-TDB, N ¼ 4;trastuzumab, N ¼ 3.

Figure 4. Efficient killing of HER2þ

cancer cells refractory to anti-HER2 therapies. A, LDH releaseassay, effectors PBMC, E:T ratio10:1, time point of 19 hours.Parental and T-DM1–resistantB and C, BT474-M1 clones weretreated with T-DM1 for 3 days (B)and cell viability was measuredusing the CellTiter-Glo or withHER2-TDB (C) for 24 hours(effectors CD8þ T cells, E:T ratio3:1, time point of 24 hours, FACSassay). Data points in the figurerepresent the mean of threesamples; error bar, SD.






HER2-TDB anti–PD-L1 combination is effective intreatment of established CT26-HER2 tumors

In the next experiment using CD3-TG mice, a similar tran-sient but significant response was seen with the HER2-TDB. In

contrast to previous study, we also detected two completeresponses (Fig. 7C). Tumor growth was significantly slower inboth of the single agent cohorts compared with the controlmice, and combination of HER2-TDB with PD-L1 blockade

Figure6. HER2-TDB inhibits growth of HER2þ tumors. A, in vivo efficacy of HER2-TDB was tested in NOD/SCID mice. A total of 5� 106 MCF7-neo/HER2cells were injected together with 1 � 107 unstimulated huPBMC from two healthy donors (PBMC 1 and 2). Mice (N ¼ 5–10) were treated with0.5 mg/kg i.v. doses of HER2-TDB on days 0, 7, and 14. Red, mice terminated before study ended; gray, mice remaining on study to study end;solid black line, fitted tumor volume for each treatment group; dashed blue line, fitted tumor volume for control group. B, MMTV-huHER2transgenic animals with established mammary tumors were treated with 0.5 mg/kg 4D5/2C11-TDB (N ¼ 7; red; mCD3-reactive 2C11 surrogate arm;weekly � 5, i.v., starting on day 0) or vehicle (N ¼ 7; black). C, progression of MMTV-huHER2 transgenic tumors and maximum percentage of tumorshrinkage by HER2-TDB treatment. D, 4D5/2C11-TDB is effective in treatment of large (>1,000 mm3) MMTV-huHER2 transgenic tumors (dosingdescribed in B). E, MMTV-huHER2 transgenic tumors growth is not affected by control TDBs in which the CD3 arm was switched to human CD3-specific(4D5/SP34-TDB; blue; N ¼ 6) or target arm to irrelevant (CTRL/2C11-TDB; gray; N ¼ 5). F, in vivo efficacy of HER2-TDB in huCD3 transgenicmice. Established CT26-HER2 tumors were treated with vehicle (N ¼ 7) or with 0.5 mg/kg HER2-TDB (4D5/SP34-TDB; every week � 3, i.v., starting onday 0; N¼ 8). G, in vivo efficacy of HER2-TDB with mCD3-reactive 2C11 surrogate arm (4D5/2C11-TDB) was tested in Balb/c mice. Dosing as describedabove (F). 15 mg/kg TDM-1 was dosed every week � 3, i.v.. Control TDB is 2C11 paired with irrelevant target arm. N ¼ 12/group.

Junttila et al.





further improved the response (Fig. 7C). Combination treat-ment resulted in durable responses; 60% of the mice livedtumor free until the study was terminated at 80 days after thefirst dose (not shown). In a repeat study (Fig. 7D), all miceresponded to the combination, 82% with complete responses,and tumor growth was controlled by the treatment in all butone mouse in the combination cohort. In summary, combi-nation of HER2-TDB with anti–PD-L1 immune therapyresulted in enhanced inhibition of tumor growth, increasedresponse rates, and durable responses.

DiscussionTDB expression and purification yields were approximately

5 mg/L. These production levels are comparable with previ-ously reported results (16) and similar to the parental anti-bodies. By optimizing strain and growth conditions, yieldssufficient for clinical studies can be obtained. The TDB formatretains binding to FcRn receptors (25), which provides a longhalf-life for the molecule and makes weekly or less frequentdosing schedule attainable. The minor alterations to theantibody that drive the heterodimerization in the knobs-into-holes technology are buried in the CH3–CH3 interactioninterface and thus are unlikely to be immunogenic. In supportof this view, only minimal and transient antitherapeutic anti-body (ATA) responses were detected in onartuzumab-treatedpatients, without apparent effect on PK profiles (34). This is astriking contrast to the experience with ertumaxomab. Ertu-

maxomab is a HER2-CD3 bispecific mouse IgG2a/rat IgG2bantibody that has been tested in a phase I study demonstratingpromising responses. Roughly one third of patients developedantibodies against ertumaxomab (HAMA/HARA) after threedoses (35).

The potency of HER2-TDB is consistently in the lowpicomolar to femtomolar range. Furthermore, as few as 10to 500 HER2-bound TDBs were sufficient to induce significantin vitro cytotoxicity, which is in agreement with what has beendescribed for the receptor occupancy required to trigger Tcell–mediated responses (29–31). With this in mind, it is notsurprising that the activity of HER2-TDB was not limited toHER2-overexpressing cells. As a result, on-target T-cell activ-ity on low expressing cells introduces an apparent safetyconcern for HER2-TDB, because HER2 is expressed in lowlevels in normal tissues, including mammary gland, kidney,and heart (36). However, our studies demonstrate a clearcorrelation between target expression levels and in vitrosensitivity to HER2-TDB. This suggests that tumor overex-pression of HER2 may provide a therapeutic index for HER2-TDB. Therefore, it may be possible to selectively target theHER2-amplified tumor cells, without affecting normal cellswhere the expression level is 10- to 100-fold lower. It isnoteworthy that the experiments in this article do not spe-cifically address safety of the HER2-TDB. Before dosinghuman patients, the safety will be addressed using extensivescrutiny and appropriate preclinical models.

Figure 7. PD-1/PD-L1 signalinglimits response to HER2-TDB. A,stimulation of human T cells byTDB and target cells induces PD-1expression. B, expression of PD-L1in target cells (293) inhibits TDB-mediated T cell–killing activity. C,effect of combinationofHER2-TDB(4D5/SP34-TDB) and anti–PD-L1on growth of established CT26-HER2 tumors in huCD3 transgenicmice. TDB dosing as in Fig. 6F;anti–PD-L1 antibody (25A1) wasdosed 10 mg/kg three times aweek � 3, i.p. Control TDB bindsto huCD3 but has an irrelevanttarget arm. TTP, time to tumorprogression (2�day 0 volume).N¼12/group.D, combination ofHER2-TDB (4D5/SP34-TDB) and anti–PD-L1 antibody results in long-term complete responses intreatment of CT26-HER2 tumors inhuCD3 transgenic mice. Dosing isdescribed above (C). CR, nodetectable tumor; PR, at least 50%tumor shrinkage from day 0.






Recruitment of T-cell killing activity with HER2-TDB isdependent on HER2 expression, but independent of the HER2signaling pathway, which predicts that HER2-TDB may beefficient in treatment of tumors that are refractory to currentanti-HER2 therapies. Our results suggest that switching toalternative mechanism of action by using HER2-TDB maybroadly enable overcoming resistance to antibody–drug con-jugates (e.g., T-DM1), targeted small-molecule inhibitors (e.g.,lapatinib), and therapeutic monoclonal antibodies that blockthe pathway signaling (e.g., trastuzumab).We also discovered apotential general resistance mechanism for T cell–recruitingmolecules with important diagnostic impact. The finding thatPD-L1 expressed by the tumor cells can inhibit the activity ofHER2-TDB also provides a mechanistic rationale for combi-nation with PD-L1 blockade.

Taken together, this study presents a new immune therapyfor HER2þ breast cancer with an alternative, extremely potentmechanism of action that is broadly effective in cells resistantto current HER2-targeted therapies. Several significantadvances are provided to bispecific T cell–recruiting antibo-dies. We (i) characterize a critical resistance mechanism, (ii)discover a potential diagnostic, (iii) introduce a novel huCD3transgenic efficacy model, and (iv) significantly improve thedrug-like properties by using technology based on full-lengthantibodies with a natural architecture. Finally, we demonstratethe benefit of combining two immune therapies: direct poly-clonal recruitment of T-cell activity together with inhibiting

the T cell–suppressive PD-1/PD-L1 signaling results inenhanced and durable long-term responses.

Disclosure of Potential Conflicts of InterestAll authors have ownership interest in Roche.

Authors' ContributionsConception and design: T.T. Junttila, J. Li, B.-E. Wang, G. Li, A. Polson,J.M. Scheer, M.R. Junttila, R. Kelley, K. Totpal, A. EbensDevelopment of methodology: T.T. Junttila, J. Li, B.-E. Wang, Y. Li, G. Li,J. Young, G.L. Phillips, B. Irving, J.M. Scheer, M.R. JunttilaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T.T. Junttila, J. Li, J. Johnston, M. Hristopoulos,R. Clark, M. Mathieu, B.-E. Wang, J. Young, E. Luis, E. Stefanich, C. Spiess,M.R. Junttila, M.S. DennisAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T.T. Junttila, J. Li, M. Hristopoulos, R. Clark, B.-E.Wang, G. Li, J. Young, E. Luis, E. Stefanich, M.R. Junttila, A. EbensWriting, review, and/or revision of the manuscript: T.T. Junttila, J. Li,D. Ellerman, G. Li, G.L. Phillips, E. Stefanich, C. Spiess, A. Polson, B. Irving, M.R.Junttila, A. EbensAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J. Li, D. Ellerman, M. Mathieu, G. Li,B. Irving, J.M. Scheer, M.R. Junttila, M.S. DennisStudy supervision: T.T. Junttila, M.R. Junttila, K. TotpalOther (designed and supervised generation of testmolecules):M.S. Dennis

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 7, 2014; revised April 3, 2014; accepted May 9, 2014;published OnlineFirst September 16, 2014.

References1. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen

JB, et al. Improved survival with ipilimumab in patients with metastaticmelanoma. N Engl J Med 2010;363:711–23.

2. Chen DS, Mellman I. Oncology meets immunology: the cancer-immu-nity cycle. Immunity 2013;39:1–10.

3. Bargou R, Leo E, Zugmaier G, Klinger M, Goebeler M, Knop S, et al.Tumor regression in cancer patients by very low doses of a T cell–engaging antibody. Science 2008;321:974–7.

4. Holmes D. Buy bispecific antibodies. Nat Rev Drug Discov 2011;10:798–800.

5. Frampton JE. Catumaxomab: in malignant ascites. Drugs 2012;72:1399–410.

6. Baselga J, Cortes J, Kim SB, Im SA, Hegg R, Im YH, et al. Pertuzumabplus trastuzumab plus docetaxel for metastatic breast cancer. N EnglJ Med 2012;366:109–19.

7. Joensuu H, Kellokumpu-Lehtinen PL, Bono P, Alanko T, Kataja V,Asola R, et al. Adjuvant docetaxel or vinorelbine with or with-out trastuzumab for breast cancer. N Engl J Med 2006;354:809–20.

8. Junttila TT, Tanner M, Isola J. [Targeted cytotoxic drugs emerging forcancer therapy]. Duodecim 2011;127:343–9.

9. Verma S, Miles D, Gianni L, Krop IE, Welslau M, Baselga J, et al.Trastuzumab emtansine for HER2-positive advanced breast cancer.N Engl J Med 2012;367:1783–91.

10. Junttila TT, Akita RW, ParsonsK, FieldsC, Lewis PhillipsGD, FriedmanLS, et al. Ligand-independent HER2/HER3/PI3K complex is disruptedby trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941. Cancer Cell 2009;15:429–40.

11. Nahta R, Yu D, Hung MC, Hortobagyi GN, Esteva FJ. Mechanisms ofdisease: understanding resistance toHER2-targeted therapy in humanbreast cancer. Nat Clin Pract Oncol 2006;3:269–80.

12. Stern HM. Improving treatment of HER2-positive cancers: opportu-nities and challenges. Sci Translational Med 2012;4:127rv2.

13. Carter P, Presta L, Gorman CM, Ridgway JB, Henner D, Wong WL,et al. Humanization of an anti-p185HER2 antibody for human cancertherapy. Proc Natl Acad Sci U S A 1992;89:4285–9.

14. Atwell S, Ridgway JB, Wells JA, Carter P. Stable heterodimers fromremodeling thedomain interface of a homodimer using aphagedisplaylibrary. J Mol Biol 1997;270:26–35.

15. Zhu Z, Lewis GD, Carter P. Engineering high affinity humanized anti-p185HER2/anti-CD3 bispecific F(ab')2 for efficient lysis of p185HER2overexpressing tumor cells. Int J Cancer 1995;62:319–24.

16. Spiess C, Merchant M, Huang A, Zheng Z, Yang NY, Peng J, et al.Bispecific antibodies with natural architecture produced by co-cultureof bacteria expressing two distinct half-antibodies. Nat Biotechnol2013;31:753–8.

17. Leo O, FooM, Sachs DH, Samelson LE, Bluestone JA. Identification ofa monoclonal antibody specific for a murine T3 polypeptide. Proc NatlAcad Sci U S A 1987;84:1374–8.

18. Pessano S, Oettgen H, Bhan AK, Terhorst C. The T3/T cell receptorcomplex: antigenic distinction between the two 20-kd T3 (T3-delta andT3-epsilon) subunits. EMBO J 1985;4:337–44.

19. Jackman J, Chen Y, Huang A, Moffat B, Scheer JM, Leong SR, et al.Development of a two-part strategy to identify a therapeutic humanbispecific antibody that inhibits IgE receptor signaling. J Biol Chem2010;285:20850–9.

20. Ramirez-Carrozzi V, SambandamA, Luis E, Lin Z, Jeet S, Lesch J, et al.IL-17C regulates the innate immune function of epithelial cells in anautocrine manner. Nat Immunol 2011;12:1159–66.

21. Junttila TT, Parsons K, OlssonC, Lu Y, Xin Y, Theriault J, et al. Superiorin vivo efficacy of afucosylated trastuzumab in the treatment of HER2-amplified breast cancer. Cancer Res 2010;70:4481–9.

22. Finkle D, Quan ZR, Asghari V, Kloss J, Ghaboosi N, Mai E, et al. HER2-targeted therapy reduces incidence and progression of midlife mam-mary tumors in female murine mammary tumor virus huHER2-trans-genic mice. Clin Cancer Res 2004;10:2499–511.


Junttila et al.




23. de laHera A,Muller U,OlssonC, Isaaz S, Tunnacliffe A. Structure of theT cell antigen receptor (TCR): two CD3 epsilon subunits in a functionalTCR/CD3 complex. J Exp Med 1991;173:7–17.

24. Jefferis R. Recombinant antibody therapeutics: the impact of glyco-sylation on mechanisms of action. Trends Pharmacol Sci 2009;30:356–62.

25. Simmons LC, Reilly D, Klimowski L, Raju TS, Meng G, Sims P, et al.Expression of full-length immunoglobulins in Escherichia coli: rapidand efficient production of aglycosylated antibodies. J Immunol Meth-ods 2002;263:133–47.

26. Aguilar Z, Akita RW, Finn RS, Ramos BL, Pegram MD, Kabbinavar FF,et al. Biologic effects of heregulin/neu differentiation factor on normaland malignant human breast and ovarian epithelial cells. Oncogene1999;18:6050–62.

27. Cameron BJ, Gerry AB, Dukes J, Harper JV, Kannan V, Bianchi FC,et al. Identification of a Titin-derived HLA-A1-presented peptide as across-reactive target for engineered MAGE A3-directed T cells. SciTranslational Med 2013;5:197ra03.

28. Ma J, Guo L, Fiene SJ, Anson BD, Thomson JA, Kamp TJ, et al. Highpurity human-induced pluripotent stem cell–derived cardiomyocytes:electrophysiological properties of action potentials and ionic currents.Am J Physiol Heart Circ Physiol 2011;301:H2006–17.

29. Irvine DJ, Purbhoo MA, Krogsgaard M, Davis MM. Direct observationof ligand recognition by T cells. Nature 2002;419:845–9.

30. Purbhoo MA, Irvine DJ, Huppa JB, Davis MM. T cell killing does notrequire the formation of a stable mature immunological synapse. NatImmunol 2004;5:524–30.

31. Sykulev Y, Joo M, Vturina I, Tsomides TJ, Eisen HN. Evidence that asingle peptide-MHC complex on a target cell can elicit a cytolytic T cellresponse. Immunity 1996;4:565–71.

32. Junttila TT, Li G, Parsons K, PhillipsGL, SliwkowskiMX. Trastuzumab-DM1 (T-DM1) retains all the mechanisms of action of trastuzumab andefficiently inhibits growth of lapatinib insensitive breast cancer. BreastCancer Res Treat 2010;128:347–56.

33. Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E,et al. Targeting HER2-positive breast cancer with trastuzumab-DM1,an antibody-cytotoxic drug conjugate. Cancer Res 2008;68:9280–90.

34. Bai S, Xin Y, Jin D, Kaur S, Nijem I, Bothos JG, et al. Populationpharmacokinetic analysis from phase I and phase II studies of thehumanized monovalent antibody, MetMAb, in patients with advancedsolid tumors. J Clin Oncol 2011;29:2571.

35. Kiewe P, Hasmuller S, Kahlert S, Heinrigs M, Rack B, Marme A, et al.Phase I trial of the trifunctional anti-HER2 x anti-CD3 antibody ertu-maxomab in metastatic breast cancer. Clin Cancer Res 2006;12:3085–91.

36. Press MF, Cordon-Cardo C, Slamon DJ. Expression of the HER-2/neuproto-oncogene in normal human adult and fetal tissues. Oncogene1990;5:953–62.






2014;74:5561-5571. Published OnlineFirst September 16, 2014.Cancer Res Teemu T. Junttila, Ji Li, Jennifer Johnston, et al. Activates T CellsAntitumor Efficacy of a Bispecific Antibody That Targets HER2 and

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