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Large Molecule Therapeutics

Bispecific Antibodies and Antibody–DrugConjugates (ADCs) Bridging HER2 and ProlactinReceptor Improve Efficacy of HER2 ADCsJulian Andreev, Nithya Thambi, Andres E. Perez Bay, Frank Delfino,Joel Martin, Marcus P. Kelly, Jessica R. Kirshner, Ashique Rafique,Arthur Kunz, Thomas Nittoli, Douglas MacDonald, Christopher Daly,William Olson, and Gavin Thurston

Abstract

The properties of cell surface proteins targeted by antibody–drug conjugates (ADCs) have not been fully exploited; ofparticular importance are the rate of internalization and theroute of intracellular trafficking. In this study, we comparedthe trafficking of HER2, which is the target of the clinicallyapproved ADC ado-trastuzumab emtansine (T-DM1), withthat of prolactin receptor (PRLR), another potential target inbreast cancer. In contrast to HER2, we found that PRLR israpidly and constitutively internalized, and traffics efficientlyto lysosomes, where it is degraded. The PRLR cytoplasmicdomain is necessary to promote rapid internalization anddegradation, and when transferred to HER2, enhances HER2degradation. In accordance with these findings, low levels ofcell surface PRLR (�30,000 surface receptors per cell) are

sufficient to mediate effective killing by PRLR ADC, whereascell killing by HER2 ADC requires higher levels of cell surfaceHER2 (�106 surface receptors per cell). Noncovalently cross-linking HER2 to PRLR at the cell surface, using a bispecificantibody that binds to both receptors, dramatically enhancesthe degradation of HER2 as well as the cell killing activity of anoncompeting HER2 ADC. Furthermore, in breast cancercells that coexpress HER2 and PRLR, a HER2xPRLR bispecificADC kills more effectively than HER2 ADC. These resultsemphasize that intracellular trafficking of ADC targets is a keyproperty for their activity and, further, that coupling an ADCtarget to a rapidly internalizing protein may be a usefulapproach to enhance internalization and cell killing activityof ADCs. Mol Cancer Ther; 16(4); 681–93. �2017 AACR.

IntroductionBreast cancer remains a disease of highunmetmedical need (1).

Monoclonal antibodies have proven to be effective therapies forthis disease, with two antibodies targeting HER2 (trastuzumaband pertuzumab) approved for treatment.

Recently, antibody–drug conjugates (ADCs) were introducedinto clinical practice with the goal of improving efficacy ofantibody-based treatments (2, 3). ADCs are designed to selec-tively kill cancer cells by combining the specificity of a monoclo-nal antibody with the intracellular delivery of a highly potentcytotoxic agent that usually targets tubulin or DNA. ADC targetsneed to be selectively expressed on tumor cells versus vital normalcells andmust be able to deliver sufficient amounts of ADC to theintended intracellular compartments to ensure efficient ADCprocessing and drug release (4).

The HER2-directed ADC, ado-trastuzumab emtansine, orT-DM1 (trastuzumab conjugated with a noncleavable linkerto tubulin inhibitor, DM1) was approved by the Food and DrugAdministration for the treatment of patients with HER2-over-expressing metastatic breast cancer who have received priortreatment with trastuzumab and a taxane (5, 6). The efficacy ofT-DM1 has been demonstrated only in patients with HER2overexpression defined as IHC3þ (7) or FISH amplificationratio � 2, which is only about half of metastatic breast cancerpatients with detectable IHC HER2 expression (8, 9). Thus,there is a need for HER2-directed ADCs that are more effectivein patients expressing low/moderate levels of HER2. Becausetargeting tissues with low HER2 expression might also induceunwanted toxicity, using bispecific antibodies (bsAb) to HER2and a target selectively expressed in breast tumors may over-come this potential limitation.

We have recently generated an ADC against such breastcancer target, prolactin receptor (PRLR), a type 1 cytokine re-ceptor that functions in mammary gland development andlactation (M.P. Kelly; unpublished data). PRLR is expressed in asubset of breast tumors (10, 11) and is implicated in thepathogenesis of breast cancer (12, 13). However, unlike HER2,PRLR is not commonly amplified in breast cancer and isgenerally expressed at much lower levels on the cell surfacethan HER2. Despite the lower levels of expression, PRLR ADC(PRLR antibody conjugated with noncleavable linker to DM1)induces efficient target-mediated killing of breast cancer cells

Regeneron Pharmaceuticals, Tarrytown, New York.

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

Corresponding Author: Julian Andreev, Regeneron Pharmaceuticals, 777 OldSaw Mill River Road, Tarrytown, NY 10591. Phone: 914-847-5433; Fax: 914-847-7544; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-16-0658

�2017 American Association for Cancer Research.

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in vitro and potently inhibits the growth of breast tumorxenografts in vivo (M.P. Kelly; unpublished data).

Because internalizedHER2 appears to predominantly recycle tothe cell surface rather than traffic to lysosomes (14, 15), wehypothesized that the difference in killing efficiency betweenPRLR and HER2 ADCs is due to the difference in lysosomaltrafficking of their targets. Indeed, we demonstrate that in breastcancer cells, PRLR, but not HER2, constitutively traffics to lyso-somes and is rapidly degraded. Consistent with this finding, weshow that noncovalently cross-linking HER2 and PRLR at theplasma membrane using HER2xPRLR bsAbs triggers HER2 deg-radation in lysosomes. NakedHER2xPRLR bsAbs augment killingof breast cancer cells by a noncompeting HER2 ADC. Further, aHER2xPRLRbispecific ADC(bsADC)kills breast cancer cellsmoreeffectively than HER2 or PRLR ADCs. Our results suggest thatcoupling an ADC target to a rapidly internalizing protein maybe a useful approach to enhance internalization and cell killingactivity of ADCs.

Materials and MethodsAntibodies, ADCs, and other reagents

PRLR antibody, a fully humanmonoclonal antibody to humanPRLR,was generated by immunizingVelocImmunemice (16, 17).HER2 antibody ("in-house trastuzumab") is a human monoclo-nal antibody with primary sequence identical to that of trastu-zumab (18, 19). PRLR antibody and HER2 antibody were pro-duced in Chinese hamster ovary cells at Regeneron.

PRLR antibody and HER2 antibody were conjugated viasurface lysines to the maytansine derivative DM1, using a non-cleavable, hetero-bifunctional linker (succinimidyl trans-4-[maleimidylmethyl] cyclohexane-1-carboxylate, SMCC; Supple-mentary Fig. S1A). The PRLR and HER2 ADCs had averagedrug-to-antibody ratios (DAR) of 3.4 and 2.4, respectively. Anisotype-matched nonbinding control ADC was prepared as de-scribed above at average DAR of 3.5.

A bsAb with HER2 and PRLR arms (HER2(T)xPRLR) wasgenerated using the "knobs-into-holes" approach (20). TheHER2(T) arm was derived from the primary sequence of trastu-zumab. The PRLR arm was derived from a fully human VelocIm-mune PRLR antibody designated H1H7672P2.

Additional HER2 and PRLR antibodies were generated inVelocImmune mice that express a single ("universal") light chain(21) to obviate the potential for heavy-light chain mispairing.HER2xPRLR bsAbs were generated as previously described (22)using HER2 antibodies that do not cross-compete with trastuzu-mab. HER2xPRLR bsADC is composed of HER2xPRLR bsAb1conjugated to maytansine derivative DM1 via a noncleavablelinker as described above with average DAR of 3.3 (see Supple-mentary Materials and Methods for details).

Recombinant human prolactin hormone (PRL) was fromR&D Systems. Cycloheximide (CHX; ref. 23), bafilomycin A1(BafA1; ref. 24), chloroquine (25), and monensin (26) werefrom Sigma. Bortezomib (27) and lactacystin (28) were fromSelleck Chemicals.

Breast cancer cell lines and transient transfectionsAll human breast cancer cell lines used in this study were

obtained from the American Type Culture Collection, authenti-cated by short tandem repeat profiling (IDEXX Bioresearch), andcultured under standard conditions. Jump-In T-REx HEK293

cells were from Thermo Fisher Scientific. Transient transfectionswere performed using pJTI R4 CMV-TO vector. To generate T47Dcell line overexpressing HER2, T47D cells were stably transfect-ed with human full-length HER2 (HER2 FL), subcloned intopRG984 plasmid, and G418 (500 mg/mL) selection was applied.All experiments were conducted with low-passage cell cultures(<passage 10).

Inducible expression of recombinant receptorsLenti-X Tet-On Advanced Inducible Expression System (Clon-

tech) was used to generate HEK293 cells expressing either full-length PRLR (PRLR FL), HER2 FL, or HER2-PRLR chimera con-structs in a tetracycline-controlled fashion. HEK293 cells werecotransduced with lentiviruses derived from the pLVX-Tet-Onadvanced regulator vector and the pLVX-Tight-Pur-GOI responsevector with subsequent selection using G418 and puromycin, assuggested by the manufacturer.

Jump-in T-REx HEK293 cells were generated to express eitherPRLR FL or PRLR truncation mutant lacking the cytoplasmicdomain in a tetracycline-controlled fashion. The Jump-In T-RExHEK293 cells containing pre-engineered R4 site and stably expres-sing the tetracycline repressor protein were transfected with thedesired expression construct followed by 14 to 21 days of anti-biotic selection. Cell pools were expanded and analyzed forgene expression using RT-PCR.

Recombinant receptor expression was induced by doxycycline(0.7 mg/mL) for 24 hours unless indicated otherwise.

Flow cytometryBinding of PRLR andHER2 antibodies to wild-type and recom-

binant HEK293 cells was evaluated by flow cytometry as follows:cells were blocked in PBS/BSA on ice, left unstained, or incubatedwith PRLR or HER2 antibodies, followed by Alexa Fluor 488–conjugated goat anti-Human IgG Fab Fragment (Jackson Immu-noResearch Laboratories). Alternatively, cellswere incubated onlywith Alexa Fluor 488–conjugated goat anti-Human IgG FabFragment ("Secondary Ab only"). Samples were analyzed on theAccuriC6 Cytometer using FlowJo X10.0.07 software. HER2 orPRLR surface levels were expressed as F/B ratio: Fold change inmean fluorescence intensity above Background ("Secondary Abonly").

For Quantitative Flow Cytometry, PRLR and HER2 antibodieswere conjugated to R-Phycoerythrin (R-PE) using standard pro-tocols.QuantumR-PEMESF kit (Bangs Laboratories)was used forthe quantitation of PE fluorescence intensity in Molecules ofEquivalent Soluble Fluorophore (MESF). All samples were ana-lyzed on the AccuriC6Cytometer using FlowJo X10.0.07 software.Fluorescence to protein ratio (F/P) was determined for PE-con-jugated PRLR and HER2 antibodies using Simply Cellular stan-dard beads (Bangs Laboratories). The number of surface receptorsper cell was determined by dividing MESF value at saturatingconcentration of an antibody by the value of respective F/P ratio.

Direct cell count–based cytotoxicity assay and cell-cycle arrestmeasurements

Tumor cells were grown in 96-well CellCoat optical plates(Greiner) at 37�C with 5% CO2 overnight. ADCs were added tocells at indicated concentrations in triplicates. For direct cellcount–based cytotoxicity assay, cells were incubated with ADCfor 3 days (unless indicated otherwise) and then fixed, permea-bilized, and nuclei-stained in one step, as described in ref. 29.

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Whole well images were acquired on automated microscopeImageXpressMICRO and analyzed for total nuclei count usingMetaXpress software (MD). Cell viability was determined as thepercentage of Hoechst DNA stain–labeled nuclei for ADC-treatedcells relative to untreated cells. IC50 values were calculated usingGraphPad Prism software.

Cell-cycle arrest was measured using increase in percentage ofearly mitotic cells as detected with anti–phospho-Histone H3(Ser10) antibody, which labels cells undergoing chromosomecondensation during mitosis (30). Twenty-four hours followingADC treatment, cells were fixed with 4% PFA, permeabilized with0.1% Triton X-100, and stained with Alexa Fluor 647–labeledanti–phospho-HistoneH3 (Ser10; CST) antibody solution con-taining Hoechst33342. Whole well images were acquired andanalyzed using MetaXpress software.

Trafficking assaysPRLR or HER2 antibodies were conjugated with the fluoro-

phores CF594 (Biotium) or Alexa Fluor 405 or with pHrodo(Thermo Fisher Scientific) according to the manufacturer'sinstructions. Noninternalized primary antibodies on the cellsurface were detected with Alexa Fluor 488–conjugated goatanti-human IgG Fab Fragment (Jackson ImmunoResearch Labo-ratories). Lysosomes were labeled by incubating cells with fluo-rescein-3 kDa dextrans for 1 hour. Nuclei were stained withHoechst 33342. Epifluorescence images were acquired on Ima-geXpressMICRO at 40x. Confocal images were acquired on ZeissLSM780 at 40x. Colocalization was quantified in a pixel-by-pixelbasis for all confocal sections of the confocal stack. The Menders'coefficient was used (31). Microscopy image quantifications wereperformed with Zen software (Zeiss; see SupplementaryMaterialsand Methods for additional details).

Surface plasmon resonanceThe affinities of PRLR or HER2 antibodies for human PRLR

or HER2 were measured using a Biacore T200 (GE Healthcare;see Supplementary Materials and Methods for additionaldetails). Kinetic parameters were obtained by globally fittingthe data to a 1:1 binding model using Biacore T200 EvaluationSoftware. The equilibrium dissociation constant (KD) was cal-culated by dividing the dissociation rate constant (kd) by theassociation rate constant (ka).

Western blottingCHX was used at 50 mg/mL. Cell lysates (RIPA buffer) were

resolved on 4% to 20% Novex Tris-Glycine gels and blotted toPVDF membranes (Novex). The following antibodies were usedfor primary labeling of membranes: PRLR antibodies (Life Tech-nologies; 35-9200), HER2 antibodies (DAKO; A0485), a-tubulinantibodies (Sigma Aldrich; T5168), b-actin antibodies (SantaCruz Biotechnology; sc-47778), and GAPDH antibodies (Gene-Tex;GTX100313). Labelingwithprimary antibodieswas followedby horseradish peroxidase–conjugated secondary antibody (GEHealthcare) at 1:5,000 and chemiluminescence detection.

ResultsPRLR, but not HER2, is constitutively internalized and trafficsefficiently to lysosomes

Previous reports indicate that certain class 1 cytokine receptors,including PRLR, undergo rapid internalization (32–34). We rea-

soned that such receptors might be better ADC targets at lowsurface expression levels than proteins that do not rapidly andconstitutively internalize, such as HER2. To measure PRLR andHER2 internalization, fluorescently labeled PRLR or HER2antibodies were bound to T47D breast cancer cells at 4�C andallowed to internalize at 37�C for varying times. Internalizationof PRLR antibody was evident by the intracellular accumula-tion of primary antibodies, whereas HER2 antibody remainedon the cell surface (Fig. 1A, top and bottom). As shown on thegraph in Fig. 1A, about 90% of PRLR antibody was internalizedwithin 60 minutes, whereas minimal internalization was observ-ed forHER2 antibody. The PRLR antibody used in this experimentblocks PRL binding and signaling (10), indicating that PRLpresent in the medium is not required for internalization.

Upon internalization into early endosomes, antibodies andtheir targets may follow a recycling pathway leading throughrecycling endosomes back to the cell surface, and/or a lysosomalpathway leading through late endosomes to lysosomes (35). Toevaluate lysosomal trafficking of PRLR and HER2, we measuredthe kinetics bywhichdextran-labeled lysosomeswere occupied byinternalized fluorescently labeled PRLR or HER2 antibodies.Confocal images showed PRLR antibody signal progressivelymoved away from the plasma membrane into dextran-labeledlysosomes, whereas the HER2 antibody signal remained in theplasma membrane (Fig. 1B, top and bottom). After 70 minutes,PRLR antibody occupied approximately 80% of the lysosomalcompartment, whereas HER2 antibody occupied approximately20%of the lysosomal compartment (graph in Fig. 1B).Upon evenlonger incubation times (24 hours), PRLR, but not HER2 anti-body, accumulated in low pH vesicles, as detected using the pH-sensitive marker pHrodo (ref. 36; Supplementary Fig. S1B). Thisobservation provides further confirmation that PRLR, but notHER2, traffics to the lysosomal compartment.

Low levels of cell surface PRLR, but not HER2, are sufficientto mediate ADC cell killing in vitro

To determine if the dramatic differences in internalizationrates and lysosomal trafficking of PRLR and HER2 wouldtranslate into differences in ADC-mediated cell killing, wecompared the efficacy of PRLR and HER2 ADCs in breast cancercells expressing high or low surface levels of PRLR and HER2. Asshown in Fig 2A, breast cancer cell line SK-BR-3 expressed highsurface levels of HER2 (1.19 � 106 receptors per cell), and verylow surface levels of PRLR (1.04 � 104 receptors per cell). Asexpected, HER2 ADC induced robust killing of SK-BR-3 cells(0.2 nM IC50), whereas PRLR ADC had minimal effect on cellviability, similar to nonbinding control ADC. In contrast, theT47D breast cancer cell line expressed a relatively low surfacelevel of HER2 (4.00 � 104 receptors per cell) and an even lowersurface level of PRLR (2.73 � 104 receptors per cell; Fig. 2B).Consistent with the rapid internalization and degradation ofPRLR, T47D cells were sensitive to PRLR ADC–induced cellkilling (0.4 nM IC50), but were completely resistant to HER2ADC. Importantly, ADCs generated using other PRLR antibodieswere similarly effective in T47D cells (not shown).

To assess whether increasing HER2 surface expression inT47D cells would increase their susceptibility to killing byHER2 ADC, we generated a T47D cell line stably overexpressingHER2 (T47D/HER2). As shown in Fig. 2C, similar to the T47Dparental cell line, T47D/HER2 cells expressed low PRLR surfacelevels (3.41 � 104 receptors per cell) and were sensitive to PRLR

HER2xPRLR Bispecific ADCs Improve Upon HER2 ADC Efficacy

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Figure 1.

PRLR, but not HER2, undergoes rapid, constitutive lysosomal internalization. Confocal microscopy images of T47D cells showing PRLR or HER2internalization (A) and lysosomal trafficking (B) were taken at different time points. For each time point, two representative confocal sections (perinuclearand supranuclear) and the XZ view are shown. A, PRLR or HER2 primary antibodies were labeled with CF594 (red); noninternalized primary antibodieson the cell surface were stained with Alexa Fluor 488–conjugated secondary antibodies (green). Scale, 10 mm. A, graph. Internalization of PRLR andHER2 was quantified as the percentage of primary antibody colocalized with secondary antibody at a particular time point (% surface receptor),mean � StErr. B, PRLR or HER2 primary antibodies were labeled with CF594 (red), lysosomes were labeled with fluorescein-3 kDa dextrans (green). Scale,10 mm. B, graph. Colocalization of PRLR or HER2 with lysosomes over time was quantified as the percentage of 3 kDa dextrans colocalized withreceptor antibody (% Lysosomes colocalizing with internalized receptor; mean � StErr).

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ADC (0.5 nM IC50). Contrary to the T47D parental cell line,surface levels of HER2 were intermediate in T47D/HER2 cells(2.07 � 105 receptors per cell), and a certain degree of sensi-tivity to HER2 ADC–mediated killing was restored (40 nMIC50). Taken together, these data confirmed previously pub-lished observations that high HER2 surface levels are requiredto mediate HER2 ADC activity (37). In contrast, and consistentwith our intracellular trafficking studies, relatively low surfacelevels of PRLR are sufficient to mediate robust ADC killing oftumor cells in vitro (IC50 < 1 nM).

PRLR, but not HER2, is rapidly degraded in lysosomesMultiple studies have shown that antibodies can promote

internalization and lysosomal trafficking of their target recep-tors (38–40). In order to investigate the turnover of PRLR andHER2 in the absence of their respective antibodies, proteinturnover in T47D cells was evaluated in a CHX chase assay. Asshown in Fig. 3A, inhibition of protein synthesis with CHX hadlittle effect on HER2 levels, whereas PRLR degradation wasfirst evident 60 minutes after CHX addition and was almostcomplete after 120 minutes. These results indicate that PRLR,but not HER2, undergoes rapid turnover in T47D cells in theabsence of antibodies.

To study PRLR turnover in a different cell system, HEK293cells induced to express PRLR were preincubated with eitherdimethyl sulfoxide (DMSO) or with the lysosomal inhib-itors BafA1, chloroquine, or monensin, and subjected to CHXtreatment for different times. The results of this experiment(Fig. 3B) demonstrated that overexpressed PRLR is rapidlydegraded in HEK293 cells with kinetics similar to thoseobserved in T47D cells and that lysosomal inhibitors chloro-quine and monensin completely abrogated PRLR degradationin the CHX chase assay, whereas another lysosomal inhibitor,BafA1, strongly reduced PRLR degradation. In contrast, theproteasome inhibitors bortezomib and lactacystin had littleor no effect on turnover of overexpressed PRLR (Supplemen-tary Fig. S2), suggesting a lack of proteasome involvement.Moreover, PRLR degradation in the CHX chase assay was notsignificantly affected by addition of PRLR antibody or PRL(Supplementary Fig. S3), further indicating that constitutive

PRLR turnover is largely independent of PRL and PRLRantibody.

To evaluate the role of lysosomal processing in PRLR andHER2ADC–mediated cell-cycle arrest, we treated T47D cells with dif-ferent concentrations of PRLR or HER2 ADCs in the presence orabsence of BafA1. We found that PRLR, but not HER2 ADC,increased the percentage of early mitotic T47D cells in a dose-dependent manner (Fig. 3C). This effect was completely abol-ished by coincubation with BafA1, indicating that acidic lyso-somal pH is needed for PRLR ADC–mediated cell-cycle arrest.Thus, rapid constitutive internalization of cell surface PRLR mayallow for efficient accumulation of PRLR ADC in lysosomes,supplying enough catabolized lysine-MCC-DM1 (41) to triggercell-cycle arrest and cell death.

PRLR turnover is mediated by its cytoplasmic/transmembranedomain and is required for ADC efficiency

To identify domain(s) of PRLR that confer efficient traffic-king to lysosomes, receptor chimeras consisting of extracellular(ecto), transmembrane (TM), and cytoplasmic (cyto) domainsof PRLR and HER2 were constructed (Fig. 3D).

To study internalization of the receptor chimeras, HEK293cells expressing either PRLR FL, HER2 FL, HER2ectoPRLRcy-toTM, or PRLRectoHER2cytoTM were transiently transfectedin 293 cells, and subjected to ice-to-37�C internalization assaywith either HER2 or PRLR antibodies. As shown in Fig. 3E,overexpressed PRLR FL, but not HER2 FL, completely inter-nalized within 1 hour. Importantly, PRLRectoHER2cytoTM didnot internalize, whereas HER2ectoPRLRcytoTM completelyinternalized within 1 hour, indicating that the PRLR transmem-brane and cytoplasmic domains are essential for rapid inter-nalization of the receptor.

To demonstrate the importance of the PRLRcytoTM in medi-ating the effect of PRLR ADC on cell-cycle arrest, T-REx HEK293cells were induced with different doxycycline concentrationsto express different surface levels of PRLR, HER2, or PRLR-HER2 chimeras (Fig. 3F). As shown in Fig. 3F, PRLR or HER2ADC–induced cell-cycle arrest positively correlated with surfacelevels of the expressed receptors. However, HER2 ADC requir-ed at least 10-fold more HER2 on the surface of HEK293 cells

Figure 2.

Low levels of surface PRLR, but not HER2, are sufficient to mediate ADC cell killing in vitro. SK-BR-3 (A), T47D (B), or T47D/HER2 (C) cells were treated withdifferent concentrations of indicated ADCs for 3 days, processed for cell cytotoxicity assay, and IC50 values were calculated. The experiment wasdone in triplicate, mean � StErr. Numbers of PRLR and HER2 surface receptors were determined by quantitative flow cytometry.






Figure 3.

PRLR, but not HER2, is rapidly degraded in lysosomes, which is essential for ADC-mediated cell-cycle arrest. A, T47D cells were incubated with CHX forthe indicated times, lysed, and processed for Western blotting. B, HEK293 cells induced to express PRLR FL were pretreated for 2 hours with DMSOor BafA1 (1 mM) or chloroquine (10 mM), or monensin (10 mM). Cells were then incubated with CHX for the indicated times, lysed, and processedfor Western blotting. C, T47D cells were treated for 24 hours with different concentrations of indicated ADCs in the absence or presence of BafA1 (1 mM),and the cell-cycle arrest was evaluated. The experiment was done in triplicate, mean � StErr. D, Schematic representation of PRLR, HER2, andPRLR-HER2 chimeric receptors. Full-length PRLR and HER2 are termed PRLR FL and HER2 FL. (Continued on the following page.)

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to induce cell-cycle arrest similar in magnitude to that inducedby PRLR ADC (Fig. 3F, left). PRLR ADC was not efficient incells expressing PRLRectoHER2cytoTM (Fig. 3F, left), whereasHER2 ADC caused efficient cell-cycle arrest in cells expressingHER2ectoPRLRcytoTM (Fig. 3F, right).

Taken together, these data demonstrate that the cytoplasmic/transmembrane domain of PRLR is not only responsible forrapid internalization of the receptor (Fig. 3E), but also forefficient ADC-mediated cell-cycle arrest (Fig. 3F).

HER2(T)xPRLR bsAb exploits constitutive lysosomaltrafficking of PRLR to promote HER2 degradation

Given the distinct trafficking properties of PRLR and HER2, wehypothesized that bsAbs bridging PRLR and HER2 receptors onthe cell surface might promote lysosomal trafficking of HER2and possibly enhance the activity of HER2 ADC (Fig. 4A). To testthis hypothesis, we generated a HER2(T)xPRLR bsAb (HER2(T)xPRLR) containing a HER2 arm from trastuzumab and a PRLRarm from antibody H1H7672P2. HER2(T)xPRLR displayedhigh-affinity binding to soluble extracellular domains of bothHER2 and PRLR (Supplementary Table S1).

As shown in Fig. 4B (left), application of HER2(T)xPRLRinduced degradation of HER2, but not of PRLR. PRLR levels didnot change because it is constantly being synthesized in endo-plasmic reticulum and delivered to plasma membrane (Fig. 4A).HER2 degradation was almost complete 5 hours after HER2(T)xPRLR addition (Fig. 4B, left).Moreover, HER2(T)xPRLR effect onHER2 levels was blocked by Monensin (Fig. 4B, right), indicatingthat, similarly to PRLR turnover, HER2(T)xPRLR-induced HER2degradation is a lysosomal process. In contrast, incubation ofT47D cells with PRLR or HER2 antibodies, either separately orcombined, did not affect HER2 levels (not shown).

Importantly, no HER2 degradation was detected when bind-ing of either PRLR or HER2 arm of HER2(T)xPRLR was blockedwith the respective receptor extracellular domain (Fig. 4C),indicating that degradation of HER2 by HER2(T)xPRLR requiresbinding of the bsAb to both targets. Finally, we show that HER2(T)xPRLR promotes HER2 degradation in T-REx HEK293 cellsexpressing PRLR FL (Fig. 4D, top), but not in cells expressingtruncated PRLR lacking the entire cytoplasmic domain (Fig. 4D,bottom), indicating that both clustering of the extracellulardomains of PRLR and HER2 induced by HER2(T)xPRLR andPRLR turnover mediated by its cytoplasmic domain are requiredfor HER2 degradation.

HER2xPRLR bsAb1 induces lysosomal trafficking of HER2Next, we wanted to test whether lysosomal trafficking of HER2

ADC could be improved by combining it withHER2xPRLRbsAbs.However, HER2(T)xPRLR could not be used for this purposebecause its HER2 arm competes with HER2 ADC for binding toHER2. To circumvent this problem, we generated HER2xPRLR

bsAbs with HER2 arms derived from antibodies with epitopesdistinct from that of trastuzumab. Eighteen HER2xPRLR bsAbs,each containing one of six HER2 arms, and one of three PRLRarms, were generated as previously described (23). As determinedby Western blotting experiments, all 18 HER2xPRLR bsAbsinduced degradation of HER2 when added to T47D cells. Forinstance, as shown in Supplementary Fig. S4, HER2xPRLR bsAbsnumbered 1 to 5 mediated HER2 degradation with efficiencysimilar to that of HER2(T)xPRLR. HER2RxPRLR bsAb1was select-ed for further studies.

To test the ability of HER2xPRLR bsAb1 to initiate HER2lysosomal trafficking, fluorescently labeled PRLR and HER2antibodies were incubated with T47D cells at 37�C for theindicated times in the presence of either HER2xPRLR bsAb1(Fig 5, right) or nonbinding control Ab (Fig. 5, left), whereasthe lysosomes were labeled with fluorescent dextrans. Confocalimages showed that in the presence of HER2xPRLR bsAb1, butnot a nonbinding control Ab, the HER2 antibody signal pro-gressively moved away from the plasma membrane and intodextran-labeled lysosomes (Fig. 5), indicating that HER2xPRLRbsAb1 is capable of inducing lysosomal trafficking of HER2.HER2xPRLR bsAb1 also induced colocalization of HER2with PRLR (Fig. 5A, right), indicating that HER2, PRLR, andHER2xPRLR bsAb1 traffic to the same lysosomes.

HER2xPRLR bsAb1 enhances HER2 ADC–induced cell-cyclearrest and cell killing, whereas HER2xPRLR bsADC killsHER2þ/PRLRþ breast cancer cells more potently than HER2ADC

Because HER2xPRLR bsAb1 was able to enhance HER2 lyso-somal trafficking, we wanted to evaluate whether HER2xPRLRbsAb1 could further increase the sensitivity of T47D/HER2 cells toHER2 ADC. To this end, we first examined the effects of differentconcentrations of HER2 ADC, with or without HER2xPRLRbsAb1, on cell-cycle arrest in T47D/HER2 cells (Fig. 6A).HER2xPRLR bsAb1 augmented the effect of HER2 ADC, with upto13%cells in earlymitosis for the combination treatment (HER2ADC at 30 nmol/L). For comparison, PRLR ADC induced cell-cycle arrest in T47D/HER2 cells with up to 13% cells in earlymitosis at 30 nmol/L ADC (Fig. 6A).

To determine if HER2xPRLR bsAb1 could augment HER2 ADCeffect on cell killing, we examined the effects of different con-centrations of HER2 ADCon viability of T47D/HER2 cells with orwithout HER2xPRLR bsAb1. As shown in Fig. 6B, T47D/HER2cells were moderately sensitive to HER2 ADC, but HER2xPRLRbsAb1 increased the potency of HER2 ADC by reducing thepercentage of viable cells at lower ADC concentrations (HER2ADC þ HER2xPRLR bsAb1). As expected, PRLR ADC was highlyeffective in killing T47D/HER2 cells (0.55 nmol/L IC50).

To validate that HER2xPRLR bsAb1-mediated enhancementof cell killing by HER2 ADC was mediated by DM1 toxin, we

(Continued.) The extracellular, transmembrane, and cytoplasmic domains of PRLR or HER2 are abbreviated as Ecto, TM, and Cyto; chimeric receptorsare termed accordingly. E, HEK293 cells were transiently transfected with indicated constructs, and receptor internalization was assessed asdescribed in Materials and Methods. Representative images are shown. Scale, 10 mm. F, Left, HEK293 cells engineered to express indicated constructswere induced for 24 hours with different concentrations of doxycycline to achieve different expression levels of the receptors. Receptor surfaceexpression levels were determined by flow cytometry and expressed as F/B ratio. PRLR ADC (1 nM) was added to cells expressing PRLR FL orPRLRectoHER2cytoTM, and HER2 ADC (1 nM) was added to cells expressing HER2 FL for an additional 24 hours, followed by cell-cycle analysis.Right, HEK293 cells engineered to express indicated constructs in a tetracycline-controlled fashion were treated and processed as described abovefor F (left panel). The experiments were done in triplicates, mean � StErr.






examined DNA integrated intensity of individual nuclei ofthe same cells used to generate the data in Fig 6B. Supple-mentary Fig. S5 shows that HER2xPRLR bsAb1 increased HER2ADC–induced cell-cycle arrest in G2–M phase at 10 nM, 5 nM,and 0.5 nM of ADC. Similar to HER2xPRLR bsAb1, otherHER2xPRLR bsAbs enhanced HER2 ADC–mediated cell-cyclearrest and cell killing in T47D/HER2 cells (not shown). In sum-mary, our data indicate that HER2xPRLR bsAbs can inducelysosomal trafficking of HER2 in T47D cells and potentiate HER2ADC–induced cell-cycle arrest and cell killing in T47D/HER2cells (Fig. 6C).

As an alternative to enhancing the effect of HER2 ADC bycombining it with naked HER2xPRLR bsAb, we generated aHER2xPRLR bsADC by conjugating HER2xPRLR bsAb1 to DM1.To determine if bridging HER2 to PRLR with HER2xPRLR bsADCcould result in enhanced ADC-mediated cell killing, we comparedthe effects of different concentrations of HER2 ADC, HER2xPRLR

bsADC, and PRLR ADC on the viability of T47D/HER2 breastcancer cells. As expected, HER2xPRLR bsADC (0.4 nM IC50)was dramatically more potent then HER2 ADC judging byreduced percentage of viable cells at lower ADC concentrations,and more potent then PRLR ADC (1 nM IC50) or HER2 ADC þPRLR ADC (0.8 nM IC50) combination (Fig. 6D).

To determine if HER2xPRLR bsADC will be more effectivethan HER2 ADC in breast cancer models expressing endo-genous levels of PRLR and HER2 (Supplementary Table S2),we compared the effects of different concentrations of HER2ADC, HER2xPRLR bsADC, and PRLR ADC on the viability ofBT-483 cells, which express 6.90 � 104 HER2 surface recep-tors per cell (intermediate-to-low HER2 levels) and approxi-mately 8.0 � 103 PRLR surface receptors per cell. In accordancewith these surface levels, HER2 ADC and PRLR ADC hadrelatively modest effects on BT-483 cell viability (1.5 nM and2.5 nM IC50, respectively; Fig. 6E). In contrast, the killing

Figure 4.

PRLR turnover is the driving force directing HER2 to lysosomal degradation when both receptors are bridged using HER2(T)xPRLR bsAbs. A, Schematicrepresentation of a bsAb, HER2(T)xPRLR, bridging HER2 with constitutively internalizing PRLR. B, T47D cells were pretreated for 1.5 hours with DMSOor monensin (10 mM), incubated with HER2(T)xPRLR (10 mg/mL) for the indicated times, lysed, and processed for Western blotting. C, HER2(T)xPRLR (10 mg/mL) or HER2(T)xPRLR (10 mg/mL) preincubated with the soluble extracellular domains of human PRLR or HER2 tagged with MycMycHis(PRLR.mmh or HER2.mmh) at 37�C for 1 hour were added to T47D cells for the indicated times. Cells were then lysed and processed for Westernblotting. D, T-REx HEK293 cells induced to express either PRLR FL (top) or truncated PRLR lacking the entire cytoplasmic domain (bottom) wereincubated with CHX combined with either HER2 antibody, HER2(T)xPRLR, PRLR antibody, or nonbinding control antibody for the indicated times, lysed,and processed for Western blotting. Note that PRLR lacking the entire cytoplasmic domain runs in multiple bands on SDS-PAGE (bottom).

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Figure 5.

HER2xPRLR bsAb1 induced lysosomal trafficking of HER2. T47D cells were labeled with fluorescein-3 kDa dextrans (green) to detect lysosomes and treatedwith CF594-labeled HER2 (red) and AF405-labeled PRLR (blue) antibodies combined with either nonbinding control antibody, 10 mg/mL (left), orHER2xPRLR bsAb1, 10 mg/mL (right), for the indicated times. For each time point, two representative confocal sections (perinuclear and supranuclear)and the XZ view are shown. Scale, 10 mm. Graph. Colocalization of HER2 with lysosomes was quantified as the percentage of 3 kDa dextranscolocalized with receptor antibody detected at a particular time point (% lysosomes colocalizing with internalized receptor, mean � StErr.)






Figure 6.

HER2xPRLR bsAb1 enhances HER2 ADC–induced cell-cycle arrest and cell killing, whereas HER2xPRLR bsADC is more effective than HER2 ADC or PRLRADC. All experiments were done in triplicates, mean � StErr. A, T47D/HER2 cells were treated for 24 hours with different concentrations of indicatedADCs, or HER2 ADC combined with 10 mg/mL HER2xPRLR bsAb1, and the percentage of early mitotic cells was determined. B, T47D/HER2 cells weretreated with different concentrations of indicated ADCs, or ADCs combined with 10 mg/mL HER2xPRLR bsAb1 for 3 days, processed for cell viability assay, andIC50 values were calculated. C, Schematic representation of a bsAb (HER2xPRLR bsAb1) enhancing HER2 ADC-mediated cell-cycle arrest and cellkilling by bridging HER2 with constitutively internalizing PRLR. HER2 ADC binds to HER2 at the trastuzumab binding site, but does not internalize efficiently.PRLR drags HER2xPRLR bsAb1, HER2, and HER2 ADC to lysosomes allowing for efficient cell killing. D, T47D/HER2 cells were treated with differentconcentrations of indicated ADCs, or combination of HER2 ADCþ PRLR ADC, processed for cell viability assay, and IC50 values were calculated. E, BT-483 cellswere treated with different concentrations of indicated ADCs, or combination of HER2 ADC þ PRLR ADC for 14 days, processed for cell viability assay,and IC50 values were calculated. Surface expression levels of PRLR and HER2 in BT-483 cells were determined by quantitative flow cytometry. F, BT-483cells were treated for 48 hours with different concentrations of indicated ADCs, or combination of HER2 ADC þ PRLR ADC, and the percentage of earlymitotic cells was determined.

Andreev et al.





effect was dramatically increased when cells were treated withHER2xPRLR bsADC judging by reduced percentage of viablecells at lower ADC concentrations. HER2 ADC þ PRLR ADCcombination (0.4 nM IC50) also increased cell killing incomparison with HER2 ADC or PRLR ADC alone but to lesserextent than HER2xPRLR bsADC (0.15 nM IC50; Fig. 6E),indicating that enhanced efficacy of HER2xPRLR bsADC inBT-483 cells may come in part from monovalent and in partfrom bivalent interaction of HER2xPRLR bsADC with HER2and PRLR. This conclusion was further supported by datain Fig. 6F, demonstrating that HER2xPRLR bsADC was moreefficient at inducing cell-cycle arrest in BT-483 cells comparedwith HER2 ADC, PRLR ADC, or HER2 ADC þ PRLR ADCcombination.

HER2xPRLR bsADC was also significantly more effective thanHER2 ADC in another HER2þ/PRLRþ breast cancer cell line,MDA-MB-361, which expresses approximately 1.52 � 105 HER2and approximately 8.0 �103 PRLR surface receptors per cell(Supplementary Table S2). Taken together, these data demon-strate that HER2xPRLR bsADC has considerably more potentkilling activity than HER2 ADC in PRLRþ/HER2þ breast cancercells, likely due to increased lysosomal trafficking of HER2xPRLRbsAb compared with HER2 antibody.

DiscussionAlthough monoclonal antibodies conjugated with tubulin

polymerization inhibitors, ado-trastuzumab emtansine (T-DM1)and brentuximab vedotin, have been approved by the FDA,much remains to be learned about how to select the best targetsfor ADCs (2, 3, 42). In breast cancers expressing high levels ofHER2, experience with T-DM1 has shown that tumor-selectiveexpression of an ADC target and its ability to deliver DM1 tolysosomes are of paramount importance (43). However, despiteits ability to effectively kill breast cancer cells expressing highlevels of HER2, T-DM1 does not efficiently kill cells expressinglow and intermediate levels of HER2, possibly because a rela-tively small amount of cell surface HER2 reaches lysosomes(14, 15). Recently, we have developed an ADC to PRLR andfound that, contrary to HER2, PRLR ADC is efficacious in vitroand in vivo at relatively low surface expression levels of PRLR(M.P. Kelly; unpublished data). In this study, we demonstratedthat rapid constitutive turnover of PRLR is the mechanismbehind PRLR ADC efficacy. Moreover, we have shown that bybridging constitutively internalizing PRLR with HER2 usingbsAbs, HER2 lysosomal degradation can be triggered and HER2ADC efficacy can be significantly improved.

Rapid constitutive turnover of PRLR may allow for rapidaccumulation of PRLR ADC molecules in lysosomes, followedby degradation of the antibody and release of high amounts oflysine-MCC-DM1 leading to cell death. Consistent with ourobservations, deGoeij and colleagues have recently demonstratedthat high turnover of cytokine receptor type 2, Tissue Factor,enabled efficient intracellular delivery of ADC (44). Tissue FactorADC effectively killed a tumor cell lines expressing as low as 2 �104 target molecules per cell (45). Taken together, our work andpublished data on Tissue Factor (44, 45) indicate that highturnover proteins selectively expressed in tumors, but not innormal vital cells, may represent attractive ADC targets.

We have shown that HER2(T)xPRLR bsAb induced rapid lyso-somal degradation of HER2 and that process required both arms

of the bsAb. Moreover, additional HER2xPRLR bsAb with HER2and PRLR arms recognizing various epitopes on HER2 or PRLRwere equally effective in degrading HER2, indicating that bsAb-mediated HER2 degradation is not dependent on particular PRLRor HER2 arms, but rather on the ability of the bsAb to bridge thetargets.

Though antibody-mediated cross-linking can trigger HER2internalization and degradation by a mechanism which is notfully understood (46), in our study, we clearly demonstrated thatwhen HER2 and PRLR were bridged using HER2(T)xPRLR, PRLRconstitutive turnovermediated by its cytoplasmic domainwas thedriving force for HER2 lysosomal degradation. Not surprisingly,when a HER2xPRLR bsAb with a non-Trastuzumab arm(HER2xPRLR bsAb1) was used, constitutive turnover of PRLRallowed for lysosomal trafficking of HER2 and significantlyenhanced HER2 ADC–induced cell-cycle arrest and cell killingin T47D/HER2 cells.

Recently, de Goeij and colleagues demonstrated that ADCpayload delivery can be improved by using HER2xCD63 bsAbs;CD63 is a protein that is described to shuttle between theplasma membrane and intracellular compartments (47). Takentogether, these data and our findings provide a proof-of-con-cept for the use of high turnover surface proteins, such as PRLR,or shuttles like CD63, in combination with bsAbs, to enhanceADC efficacy.

Though a combination of HER2-ADC plus HER2xPRLRbsAb may be deployed successfully in some PRLRþ/HER2(IHC2þ) breast tumors, an alternative strategy of dual target-ing with DM1-conjugated bsAbs may work equally well oreven have advantages (48). Indeed, the HER2xPRLR bsADCwas more effective than HER2 ADC or PRLR ADC at killingbreast cancer cells that coexpress intermediate HER2 (IHC2þ)and low PRLR levels, suggesting the possibility that thisapproach could provide benefit in this subpopulation of breastcancer patients.

In summary, our study opens up the possibility of targetingconstitutively internalizing, high turnover proteins (even ifexpressed at low surface levels) with ADCs and/or of exploitinghigh turnover proteins, in combination with bsAbs, to enhancethe efficacyofADCs targetingproteins that donot efficiently trafficto lysosomes.

Disclosure of Potential Conflicts of InterestM.P. Kelly is Staff Scientist at Regeneron Pharmaceuticals. T. Nittoli has

ownership interest (including patents) in Regeneron Pharmaceuticals. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: J. Andreev, A.E. Perez Bay, F. Delfino, J. Martin,J.R. Kirshner, A. Rafique, T. Nittoli, W. Olson, G. ThurstonDevelopment of methodology: J. Andreev, A.E. Perez Bay, F. Delfino,A. Rafique, A. Kunz, T. Nittoli, G. ThurstonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Andreev, N. Thambi, A.E. Perez Bay, F. Delfino,M.P. Kelly, A. Rafique, A. KunzAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Andreev, N. Thambi, A.E. Perez Bay, F. Delfino,M.P. Kelly, A. Rafique, C. Daly, G. ThurstonWriting, review, and/or revision of the manuscript: J. Andreev, A.E. Perez Bay,A. Rafique, T. Nittoli, C. Daly, W. Olson, G. ThurstonAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. AndreevStudy supervision: J. Andreev, D. MacDonald, G. Thurston






AcknowledgmentsThe authors thank N. Papadopoulos, P. Trail, N. Stahl, and G. Yancopoulos

for helpful comments and suggestions, Wei Wang for bioinformatics analysis,and all Regeneron employees that contributed to the generation of antibodiesused in this study.

Grant SupportThis work was supported by Regeneron Pharmaceuticals, Inc.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 4, 2016; revised December 26, 2016; accepted January 3,2017; published OnlineFirst January 20, 2017.

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2017;16:681-693. Published OnlineFirst January 20, 2017.Mol Cancer Ther Julian Andreev, Nithya Thambi, Andres E. Perez Bay, et al. ADCsBridging HER2 and Prolactin Receptor Improve Efficacy of HER2

Drug Conjugates (ADCs)−Bispecific Antibodies and Antibody

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