neutralizationofprolactinreceptorfunctionbymonoclonal...

Large Molecule Therapeutics

Neutralization of Prolactin Receptor Function by MonoclonalAntibody LFA102, a Novel Potential Therapeutic for theTreatment of Breast Cancer

Jason S. Damiano, Katherine G. Rendahl, Christopher Karim, Millicent G. Embry, Majid Ghoddusi,Jocelyn Holash, Abdallah Fanidi, Tinya J. Abrams, and Judith A. Abraham

AbstractNumerous lines of evidence suggest that the polypeptide hormone prolactin (PRL)may contribute to breast

and prostate tumorigenesis through its interactions with the prolactin receptor (PRLR). Here, we describe the

biologic properties of LFA102, a humanized neutralizing monoclonal antibody directed against the extracel-

lular domain of PRLR. This antibody was found to effectively antagonize PRL-induced signaling in breast

cancer cells in vitro and in vivo and to block PRL-induced proliferation in numerous cell line models, including

examples of autocrine/paracrine PRL activity. A single administration of LFA102 resulted in regression of

PRL-dependent Nb2-11 tumor xenografts and significantly prolonged time to progression. Finally, LFA102

treatment significantly inhibited PRLR signaling as well as tumor growth in a carcinogen-induced, estrogen

receptor-positive rat mammary cancer model as a monotherapy and enhanced the efficacy of the aromatase

inhibitor letrozole when administered in combination. The biologic properties of LFA102, elucidated by

the preclinical studies presented here, suggest that this antibody has the potential to be a first-in-class, effective

therapeutic for the treatment of PRL-dependent cancers. Mol Cancer Ther; 12(3); 295–305. �2012 AACR.

IntroductionProlactin (PRL) was initially described in the 1930s as a

pituitary-derived polypeptide hormone that had thecapacity to stimulate mammary gland development andlactation, but it was not until the 1970s that PRL was firstrecognized as having the ability to contribute to theinitiation and promotion of mammary tumors in rodents.More recently, high circulating levels of PRL in humanshave been correlated with an increased risk for develop-ing breast cancer, especially estrogen receptor (ER)-pos-itive (ERþ) disease (1). Pharmacologic agents that areknown to increase systemic PRL are also associated withan increased risk for breast cancer (2). Further pointing tothe tumorigenic potential of PRL, transgenic expression ofthis hormone in mouse mammary glands induced intrae-pithelial neoplasms and invasive tumors (3). Biologic andmolecular characterization studies suggested that themajority of these tumors (irrespective of ER status) mostclosely resembled estrogen-insensitive human tumors ofthe luminal subtype, suggesting that PRL may contribute

to tumor growth in instances where estradiol does notfunction (or no longer functions) as a mitogen (4).

PRL mediates its physiologic functions throughengagement of the extracellular domain of PRLR, a typeI cytokine receptor expressed with high prevalence inhuman breast cancer (5). PRLR lacks intrinsic kinaseactivity, but upon interaction with its ligand, the dimer-ized receptor undergoes a conformational change thatactivates other intracellular signaling partners to produceits ultimate cellular effects. In addition to human PRL(hPRL), human growth hormone (hGH) and placentallactogen are also known ligands of human PRLR (hPRLR;refs. 6, 7). The signaling pathway most often associatedwith PRLR function is the Jak2/Stat5 pathway, althoughother prosurvival and anti-apoptotic signaling cascadesare also known to be triggered in cancer cell lines, includ-ing Jak1/Stat3, phosphoinositide 3-kinase (PI3K)/Akt,and Raf/MEK/ERK (8–10). PRLR has also been observedto cross-talkwith other growth factor and steroid receptorsignaling pathways, including insulin-like growth factor-1 receptor (IGF1R), EGF receptor (EGFR), HER2, and ERa(11–15), suggesting opportunities for synergy betweenan anti-PRLR therapeutic and other targeted agents inhuman breast cancer.

Early clinical investigations of agents that act by inhi-biting PRL release from the pituitary gland (e.g., thedopamine receptor agonist bromocriptine) in the treat-ment of human breast cancer were mostly inconclusive(16, 17), potentially due to the fact that non-pituitary(dopamine receptor independent) sources of PRL synthe-sis exist, including adipose, mammary, prostate, and

Authors' Affiliation: Novartis Institutes for BioMedical Research, Emery-ville, California

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

Corresponding Author: Jason S. Damiano, Novartis Institutes for Bio-Medical Research, 4560HortonSt, Emeryville, CA94608. Phone: 510-923-3014; Fax: 510-923-4550; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-0886

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

www.aacrjournals.org 295

on August 15, 2019. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst December 27, 2012; DOI: 10.1158/1535-7163.MCT-12-0886

http://mct.aacrjournals.org/

endometrial tissues, as well as breast and prostate tumorsthemselves (18–22). The presence of tumor autocrine PRLhas recently been reported to be significantly correlatedwith either high tumor grade and/or poor prognosis inbreast, endometrial, and prostate cancers (22, 23). Thus,true systemic abrogation of PRL function would require atherapeutic capable of effectively blocking endocrine,autocrine, and paracrine PRL action within a tumor. Tothis end, mutant PRL peptide antagonists acting directlyon PRLRhave been developed and used as key preclinicaltools to investigate PRLR biology (24–27); however, sev-eral inherent liabilities of these antagonists have madetheir use as therapeutics in humans difficult (25, 28). Here,we report the development and characterization of aneutralizing monoclonal antibody against the humanPRLR, designated LFA102, which is capable of inhibitingthe biologic functions of both endocrine and autocrinePRL. LFA102 potently antagonizes PRLR signaling andfunction in vitro and inhibits the growth of PRL-sensitivecancer models in vivo, suggesting that this antibody couldhave use for the treatment of PRL-dependent neoplasmsin the clinic.

Materials and MethodsMaterials

T47D and MCF7 cells were obtained from AmericanType Culture Collection and were authenticated usingshort tandem repeat profiling. T47D-T2 cells werederived from T47D cells (obtained from EG&G MasonResearch Institute) through serial passage in immuno-compromised mice to optimize in vivo growth charac-teristics and were authenticated using single-nucleotidepolymorphism analysis. Nb2-11 cells were obtainedfrom the European Collection of Cell Cultures and wereauthenticated using DNA barcoding and DNA profil-ing. BaF3 cells were obtained from the German Collec-tion of Microorganisms and Cell Cultures and wereauthenticated using standard and multiplex PCR. Allcell lines were used fewer than 6 months after resusci-tation and were not re-authenticated. Primary humanbreast tumor specimens were obtained from MT Groupin accordance with appropriate protocols and patientconsent. Cell culture conditions and procedures forgenerating engineered cell lines are described in theSupplementary Methods. The parental mouse hybrid-oma to LFA102 was generated from mice immunizedwith recombinant extracellular domain of PRLR;LFA102 was generated by humanization of the mouseantibody using Human Engineering (HE) technology atXOMA, LLC. Sources of recombinant proteins, commer-cially available antibodies, compounds, and vehicleformulations used for in vitro and in vivo studies canbe found in the Supplementary Methods.

Fluorescence-activated cell-sorting analysis ofLFA102 binding to live cells

To evaluate the levels of PRLR expressed on the plasmamembrane and the extent of anti-PRLR antibody binding,

adherent cells were detached from tissue culture flasksusing Enzyme-Free Cell Dissociation Buffer (Gibco) orwere disaggregated from a primary tumor before stainingas described in the Supplementary Methods.

PRL competition assayAmine-reactive Alexa Fluor 647 dye (A647; Invitrogen)

was used to label recombinant hPRL per manufacturer’sdirections. T47D cells [in fluorescence-activated cell-sort-ing (FACS) buffer] were incubated with antibodies or 5mg/mL unlabeled hPRL for 45 minutes on ice. A647-PRLwas then added to a concentration of 0.5 mg/mL, and thecells were incubated for 20 minutes before washing andanalysis by FACS.

Signaling assays (Western blotting)T47D and MCF7 cells were seeded into 6-well tissue

culture plates overnight before use. BaF3/hPRLR, BaF3/hPRLR/hPRL, or dissociated primary human breast can-cer cells were seeded and used in the assays withoutovernight incubation. Cells were incubated with LFA102or human IgG1 isotype control antibody in serum-freeRPMI for 30minutes at 37�Cbefore stimulationwith hPRL(50 ng/mL) for 30 minutes. Details of cell lysate prepa-ration and antibodies used for Western blotting aredescribed in the Supplementary Methods. Experimentswere carried out in triplicate.

Cell proliferation and antibody-dependent cell-mediated cytotoxicity assays

For cell proliferation analyses, cells were seeded asindicated in the Supplementary Methods and were thentreated for 4 to 7dayswithhPRLor hGH in thepresence orabsence of anti-PRLRantibodies LFA102orMAB1167. Forantibody-dependent cell-mediated cytotoxicity (ADCC)analysis, T47D cells were cocultured with fresh humannatural killer (NK) cells, as described in the Supplemen-tary Methods. CellTiter-Glo and a luminescence counter(Wallac Trilux) were used to assess cell numbers uponcompletion of the assays. Luminescence counts fromwellscontaining untreated cells (100% viability) were used tonormalize treated samples. EC50 values (half maximalinhibitory concentration of antibody) were calculatedusing GraphPad Prism 4 software. Experiments werecarried out in triplicate.

Evaluation of pharmacodynamic effects of LFA102 inT47D-T2 tumors

T47D-T2 cells (7.5 � 106) were injected in the rightmammary fat pad of female severe combined immuno-deficient (SCID) mice. Mice with approximately 125 mm3

T47D-T2 tumors were injected i.v. with 10 mg/kg ofLFA102 or human IgG1 control antibody. Forty-eighthours later,micewere injected intraperitoneally (i.p.)with100 mg ovine PRL (oPRL) or saline (5 per group). Tumorswere collected 60 minutes post-PRL injection to evaluatephosphorylated Stat5 (p-Stat5) levels by immunohis-tochemistry (IHC) and whole blood was collected for

Damiano et al.

Mol Cancer Ther; 12(3) March 2013 Molecular Cancer Therapeutics296




serum isolation to determine LFA102 concentrations (seeSupplementary Methods).

In vivo pharmacodynamic and efficacy evaluationof LFA102 in Nb2-11-luc tumor–bearing miceFemale SCID mice were injected s.c. in the right flank

with 5 � 106 Nb2-11-luc cells. To examine the impact ofLFA102 on PRL-induced signaling Nb2-11 tumors, micewere treated in a manner similar to the T47D pharma-codynamic (PD) study, except that 20 mg of oPRL wasused instead of 100 mg (described in the SupplementaryMethods). For the in vivo efficacy evaluation, tumorswere randomized into groups based on biolumines-cence 4 days postimplant (tumor burden of approxi-mately 4 � 107 photons/s; 8 per group). A singleadministration of LFA102 (0.01–10 mg/kg) or humanIgG1 control antibody (3 mg/kg) was given i.p. Biolu-minescence imaging is described in the SupplementaryMethods. Statistical significance was determined usingthe Kruskal–Wallis one-Way ANOVA on ranks, andpairwise analysis was conducted with Dunn post hoc

test. Time to progression was defined as the day animalsrequired euthanasia for clinical signs of disease burdenand statistical significance was determined using thelog-rank test.

Evaluation of LFA102 pharmacokinetics, PD andefficacy as a single agent, and activity in combinationwith letrozole in a DMBA-induced mammary tumormodel

Mammary tumors were established and treated withLFA102 as a single agent (300mg/kg twiceweekly) beforeanalysis of tumor size, p-Stat5, and LFA102 serum levels(described in Supplementary Methods). The activity ofLFA102 and letrozole as single agents and in combinationwas assessed by enrolling animals onto the study whentumor volumes reached an average of approximately 200mm3 (n¼ 15/group). LFA102was dosed i.p. at 300mg/kg3 timesweekly. Letrozolewas administered orally 2 timesdaily at a total dose of 10 mg/kg. A partially efficaciousdose of letrozole was used to maximize the potential todetect a combination effect of the 2 agents. Statistical

Figure 1. Characterization ofLFA102 binding to PRLR-expressingcells. A, LFA102 (10 mg/mL) wasincubated with T47D, MCF7, Nb2-11,PRLR-negativemouseproBcells(BaF3), BaF3 cells expressing hPRLR(BaF3/hPRLR), or disaggregatedfresh primary human breast tumorcells before FACS analysis. Open(dotted line) histograms, nonspecifichuman IgG1 control antibodyfluorescence signal; solidhistograms, LFA102 fluorescencesignal. B, antibody–ligandcompetition was determined bypreincubating T47D cells withanti-PRLR antibody (LFA102 orMAB1167) and thenexposing cells toA647-PRL for 20 minutes beforefluorescence detection by FACS.Unlabeled hPRL was added to onesample (dark gray bar) to showspecificity of A647-PRL binding tocells.

Preclinical Development of Anti-PRLR Antibody LFA102

www.aacrjournals.org Mol Cancer Ther; 12(3) March 2013 297




analysis of tumor volumes was conducted by one-wayANOVA followed by the Tukey or Holm–Sidak post hoctest.

ResultsCharacterization of LFA102 binding to PRLR

LFA102 is a humanized IgG1-kappa antibody derivedusing mouse hybridoma and Human Engineering (HE)technology. To show binding of the antibody to PRLRexpressed in its native conformation on the surface ofcells, a FACS assaywas used. In addition to binding to thehuman ERþ luminal breast cancer cell lines T47D andMCF7, LFA102 was also found to bind to freshly isolatedprimary humanbreast cancer cells and to the rat pre-T-celllymphoma line Nb2-c11, indicating rat PRLR (rPRLR)cross-reactivity of this antibody (Fig. 1A). As a demon-stration of specificity, LFA102 did not bind to PRLR-negative BaF3 cells but did bind to BaF3 cells expressingexogenous hPRLR (BaF3/hPRLR). The degree of LFA102binding to hPRLR on each cell line correlated with PRLRexpression levels assessed by Western blotting (Supple-

mentary Fig. S1). LFA102 did not bind to cells expressingmouse PRLR (data not shown).

Epitope mapping studies determined that LFA102binds to a conformational epitope within the D2 domainof hPRLR (data not shown), which contains the putativedimerization interface of the receptor and the WSXSXmotif hypothesized to be a "molecular switch" for PRLRactivation (29). Binding within the D2 domain of PRLRalso suggests that LFA102 will bind to the majority of theisoformsof PRLRdescribedpreviously (30). LFA102 is notthought to directly interact with the D1 domain of thereceptor, which contains the majority of the ligand-bind-ing pocket (31).

To characterize the LFA102 binding mode in relation toPRL–PRLR interactions, a cell-based ligand competitionassay was developed to examine the impact of antibodybinding on the ability of Alexa647-labeled PRL (A647-PRL) to associate with PRLR. Figure 1B depicts the meanfluorescence signal measured for cells pretreated withanti-PRLR antibodies (LFA102 orMAB1167) or unlabeledhPRL (specificity control). MAB1167 significantly

A

B

T47D MCF7

p-ERK1/2

Akt

p-Stat5

PRL:LFA102:

IgG1:

- - - + + + + + + +0 0 05 0 0.05 0.1 0.5 1 50 5 0 0 5 0 0 0 0 0

- - - + + + + + + +0 0 05 0 0.05 0.1 0.5 1 50 5 0 0 5 0 0 0 0 0

ERK1/2

p-Akt

Stat5

PRL:LFA102:

IgG1:

p-Stat5

Stat5

PRL:

IgG1:LFA102:

+- + ++

+--

-

- --

Primary human breast

tumor (ER+/PR+/HER2-)

CIgG1 Control IgG1 Control + PRL

LFA102 LFA102 + PRL

Figure 2. Inhibition of PRLRsignaling by LFA102. A, inhibitionof PRL-induced signaling in T47Dand MCF7 cells. Cells werepreincubated with IgG1 controlantibody or LFA102 for 30 minutesbefore stimulation with hPRL (50ng/mL) for 30minutes and analysisof lysates by Western blotting. B,fresh primary human breast cancercells were disaggregated andtreated as above. C, LFA102inhibits PRL-induced p-Stat5 inT47D-T2 xenograft tumors. SCIDmice with T47D-T2 mammarytumors were injected with anIgG control or LFA102 antibody(10mg/kg). Forty-eight hours later,saline or oPRL was administered60 minutes before tumorcollection. Representative IHCimages of p-Stat5 immunostainingare shown (�200).

Damiano et al.





displaced fluorescent signal from cells, indicating that thebinding of this antibody influences the association ofhPRLwith hPRLR. In contrast, LFA102 had no significanteffect on A647-PRL binding to PRLR, even when saturat-ing concentrations of the antibody were added. Thus,LFA102 does not bind to PRLR in a ligand-competitivemanner.

NeutralizationofPRLRsignaling inbreast cancer celllines in vitro, primary breast tumor cells ex vivo, andT47D xenograft tumors in vivoTo assess the impact of LFA102 on PRLR signaling in

vitro, T47D and MCF7 cells were used. As determinedby Western blotting with phospho-protein–specific anti-bodies, LFA102 was found to efficiently neutralize hPRL-induced phosphorylation of Stat5 (p-Stat5), Akt (p-Akt),and p42/p44 [p-extracellular signal–regulated kinase(ERK)1/2] signaling in a concentration-dependent man-ner in T47D cells (Fig. 2A). In contrast to T47D cells,MCF7cells only exhibited clear p-Stat5 activation in response tohPRL, which was efficiently antagonized by LFA102treatment. Importantly, no induction of PRLR signalingwas seen in either cell line when treated with LFA102alone, indicating that the antibody is devoid of measur-able agonistic activity.To confirm that primary human breast tumor cells

also have the capacity for PRL-induced signaling, whichcan be abrogated with LFA102 treatment, a fresh primaryhuman breast tumor specimen (characterized as ERþ/progesterone receptorþ/HER2� and PRLRþ) was used.The dissociated tumor cells showed hPRL-inducedp-Stat5 induction (Fig. 1D), which was completely neu-tralized by LFA102 at a concentration of 10 mg/mL.To show that LFA102 can neutralize PRLR activity in a

breast carcinoma in vivo, mice with T47D-T2 tumors wereadministered a dose of LFA102 or a nonspecific humanIgG1 (control antibody), followed 48 hours later by a bolusof oPRL to stimulate PRLR (mouse PRLdoes not stimulatehPRLR) or a control injection (Fig. 2C) 1 hour beforeassessment. Tumors from mice not treated with oPRLdisplayed little to no p-Stat5, whereas mice administeredcontrol antibody before oPRL stimulation showed signif-icantly elevated p-Stat5 levels in tumors. In contrast,LFA102 treatment before oPRL stimulation resulted inno detectable intratumoral p-Stat5, showing that LFA102efficiently blocked PRLR signaling in this human breastcancer model. Serum levels of LFA102 in the treated miceranged from 30 to 56 mg/mL at the time of samplecollection (data not shown), a concentration anticipatedto be achievable in humans (32).

Abrogation of endocrine and autocrine PRL-inducedcell proliferation by LFA102To evaluate the ability of LFA102 to antagonize PRL-

inducedproliferation, T47D cellswere exposed to increas-ing concentrations of hPRL in the presence of eitherLFA102 or control antibody, and changes in viable num-berwere examined. Exposure of PRL-treated T47D cells to

LFA102, but not control antibody, completely neutralizedthe effects of PRL on cell proliferation (Fig. 3A). LFA102was able to antagonize the progrowth effects of hPRL atphysiologic levels (10–200 ng/mL) and super-physiologiclevels (up to 2000 ng/mL). An ability to antagonize PRLRin the presence of high levels of ligand may be importantfor a potential therapeutic, in that pathway inhibitionmaylead to hyper-prolactinemia (33). LFA102 was also foundto completely inhibit the survival of the PRL-dependentrat pre-T-cell lymphoma cell line Nb2-11 (Fig. 3B).

To explore the activity of LFA102 in other contexts, anengineered hPRLR-expressing and hPRL-dependent cellline (BaF3/hPRLR) was used. Both LFA102 (non–PRL-competitive) andMAB1167 (PRL-competitive) antibodiesinhibited the hPRL-dependent growth of BaF3/hPRLRcells, (EC50 ¼ 0.5 vs. 3.9 mg/mL, respectively; Fig. 3C).Interestingly, only LFA102 efficiently suppressed PRL-induced cell proliferation in the presence of higherconcentrations of this ligand (Fig. 3D), despite both anti-bodies having a similar binding affinity (KD) for recom-binant human PRLR (LFA102 ¼ 2 nmol/L, MAB1167 ¼3 nmol/L; data not shown). In addition to hPRL, hGHcan also directly bind and activate hPRLR and has beenhypothesized to play a role in breast cancer (34). There-fore, we examined the ability of LFA102 to antagonizehGH-mediated activation of PRLR in BaF3/hPRLRcells (which do not express GH receptor). In these studies,LFA102 potently neutralized the effects of hGH onhPRLR, similar to its impact on the actions of hPRL (Fig.3E). Thus, LFA102 has the capacity to block cell prolifer-ation induced by multiple PRLR ligands.

Given that extrapituitary sources of PRL include mam-mary tissue and tumors, it has become apparent thattumor cells may be exposed to autocrine or paracrinesources of PRL within their microenvironment. To assessthe ability of LFA102 to inhibit PRLR function in thesecontexts, BaF3/hPRLR cells were engineered to be depen-dent on autocrine/paracrine hPRL through the expres-sion of a hPRL transgene. This cell line, termed BaF3/hPRLR/hPRL, displays an auto-activated PRLR signalingapparatus, as measured by baseline p-Stat5 levels in theabsence of exogenously added cytokines. Incubation ofthe cells with LFA102 (but not MAB1167 or control anti-body) strongly suppressed this baseline signal (Fig. 3F)and only LFA102 was found to inhibit the growthand survival of BaF3/hPRLR/hPRL cells in proliferationassays (Fig. 3G). These results indicate that ligand-com-petitive PRLR antagonists may be inefficient at neutral-izing PRLR function in instances where local PRLconcentrations may be high, consistent with previousobservations (25).

ADCC induced by LFA102Through their interactions with Fcg receptors on

immune effector cells, antibodies of the human IgG1isotype have the potential to elicit tumor cell lysis via aprocess known as ADCC. Primary human NK cells andT47D cells (which express PRLR and Her2) were used to






assess the ADCC potential of LFA102. LFA102 was foundtomediate T47D cell killingwith a similar range of activityas the anti-Her2 antibody trastuzumab (maximum cellkilling¼ 50%;EC50¼ 0.13 and 0.02mg/mL forLFA102 andtrastuzumab, respectively; Fig. 3H). Therefore, LFA102has the capacity to wield ADCC as an additional mech-anism of action in vivo.

LFA102 pharmacokinetics and pharmacodynamics inrats

Given that LFA102 binds and neutralizes rPRLR, ratswere used for in vivoPKandPDanalyses of LFA102. In thedose ranges examined, LFA102 clearance ranged from1.45 to 0.92 mL/h/kg in male rats and 13.5 to 3.93 mL/h/kg in female rats (Supplementary Table S1). The mean

estimated half-life ranged from 1.43 to 8.99 days in malesand from 0.12 to 4.23 days in females. These resultssuggest that LFA102 has a rapid clearance mechanismin female rats that is not observed inmale rats. In contrast,the T1/2 of LFA102 in female mice was between 7.1 and8.5 days, typical for a human IgG1 antibody (35). Alikely explanation for the gender difference in LFA102PK in rats is target-mediated distribution of LFA102 tofemale rat liver, which expresses significantly more PRLRthan that found in male rat liver or livers from eithergender of mouse, cynomolgus monkey or human (ref. 36and data not shown). Consequently, we expect that inhumans, the PK of LFA102 will be similar to that foundin cynomolgus monkeys and dissimilar to that seen infemale rats.

Figure 3. LFA102 inhibits cell growth and viability in vitro. A, inhibition of PRL-induced proliferation in T47D cells. Viable cell number was assessed usingCellTiter-Glo after 96 hours of exposure to hPRL in conjunction with LFA102 or IgG1 control antibody (both at 10 mg/mL). B, inhibition of Nb2-11 cell survival.Relative number of viable Nb2-11 cells was assessed as described above. C, inhibition of BaF3/hPRLR cell survival. BaF3/hPRLR cells were incubatedwith hPRL (50 ng/mL) in conjunction with LFA102, MAB1167, or species-matched IgG1 control antibodies for 96 hours before analysis. D, LFA102 efficientlyneutralizes PRLR in the presence of high PRL concentrations. BaF3/hPRLR cells were incubatedwith antibodies (10 mg/mL) and increasing concentrations ofhPRL for 96 hours before analysis. E, LFA102 inhibits hGH-induced hPRLR activation. BaF3/hPRLR cells were treated as above except in some cases hGHwas added instead of hPRL (both at 50 ng/mL). F, blockade of autocrine PRL signaling by LFA102. BaF3/hPRLR/hPRL cells were incubatedwith antibodies inthe absence of exogenously added hPRL for 60 minutes before lysis and analysis of p-Stat5 and total Stat5 levels byWestern blotting. G, LFA102 abrogatessurvival of BaF3/hPRLR/hPRL cells. Cells were incubated with antibodies in the absence of exogenously added hPRL for 168 hours before analysis. H,induction of ADCC by LFA102. Freshly isolated human NK cells were incubated with T47D cells at a ratio of 5:1 (effectors:targets) in the presence of LFA102,trastuzumab, or IgG1control antibody. Twenty-four hours later, plateswerewashedand the relative number of adhered viable cellswas assessed. Values inA,B, C, D, E, G, and H are means (�SD) of triplicate samples; y-axis, % viable cell number relative to untreated cells or untreated cell mix (H, NKþ T47D only).

Damiano et al.





To examine the PD effects of LFA102 in the rat mam-mary gland, the antibody was administered at 50 mg/kgto compensate for the abnormally high rate of LFA102clearance in this species, before ratPRL (rPRL) stimulationand tissue collection. Similar to the T47D-T2 PD studydescribed above, LFA102 strongly antagonized rPRLRsignaling in mammary tissue (Supplementary Fig. S2).Because PRLR knockout mice are hyperprolactinemic,

suggesting the existence of a compensatory physiologicreaction to PRLR signal ablation (33), we hypothesizedthat serum PRL levels may serve as a marker of PRLRpathway inhibition by LFA102. To remove the highlyvariable influence of the estrous cycle on baselinePRL levels, ovariectomized rats were used to test thishypothesis. Rats were given daily adminstrations (days0–4) of LFA102 (50 mg/kg) or vehicle. Mean serum rPRLlevels in LFA102-treated rats were significantly elevatedon days 1 to 7, relative to controls (Supplementary Fig. S3;P < 0.05). Analysis of LFA102 levels in the serum showed apositive correlation with elevated PRL (SupplementaryFig. S4). Thesedata indicate that elevatedPRL levels couldrepresent a viable biomarker for LFA102 activity inhumans. On the basis of the results shown in Fig. 3D, itis anticipated that the peripheral effects of elevated PRL

would be adequately antagonized by LFA102 in thissituation.

Inhibition of PRLR signaling and tumor growth byLFA102 in the Nb2-11-luc rat pre-T-cell lymphomatumor model in mice

It has been recognized that human breast cancer celllines are not very dependent on PRL, likely due to the factthat bovine PRL (bPRL) found in FBS does not activatehPRLR (37, 38). Thus, human cell lines have been main-tained under conditions where initially PRL-dependentcells will ultimately not survive. As bPRL does activaterPRLR, Nb2-11 cells have maintained their PRL depen-dence during culture in FBS, and for this reason, theywereused as a mechanistic model of PRL-dependent humancancer. To examine the ability of LFA102 to inhibit PRLsignaling and tumor growth in this model, mice withestablished subcutaneous Nb2-11-luc xenografts wereused. Baseline p-Stat5 levels in these tumors were lowand variable, potentially due to the inefficiency of mousePRL (mPRL) in activating rPRLR, or to the contribution ofthe estrous cycle to systemic mPRL levels. Therefore, aninjection of oPRL was administered to stimulate intratu-moral p-Stat5 levels. LFA102 treatment efficiently blocked

Figure 4. LFA102 treatment inhibitsPRLR signaling and tumor growth inthe Nb2-11-luc xenograft model. A,LFA102 treatment inhibits PRL-induced p-Stat5 in Nb2-11-luctumors. SCID mice withsubcutaneous Nb2-11-luc tumorswere injected with an IgG1 controlantibody or LFA102 at a dose of10 mg/kg. Forty-eight hours later, aninjection of saline or oPRL wasadministered (60 minutes beforetumor collection). RepresentativeIHC images of p-Stat5immunostaining are shown (�200).B, LFA102 displays antitumoractivity against Nb2-11-lucxenograft tumors. Treatment oftumor-bearing mice with a singledose of LFA102 resulted in doseproportional efficacy (dosing timepoint indicated by arrow). Tumorbioluminescence is presented asmean � SEM (�, P < 0.05). C,representative bioluminescenceimages of mice on day 14 post-randomization, administered asingle dose of 3 mg/kg IgG1 isotypecontrol antibody (top) or 10 mg/kgLFA102 (bottom). Bioluminescenceis measured as photons/sec/cm2/steradian (p/sec/cm2/sr). D,treatment of Nb2-11-luc tumor–bearing mice with a single dose ofLFA102 significantly increased timeto progression (�, P < 0.05). Thepercentage of mice lacking clinicalsigns of disease progression in eachgroup is plotted.

A

C D

0 25 50 75 100 125 1500

20

40

60

80

100

LFA102 3 mg/kgLFA102 10 mg/kg

LFA102 1 mg/kgIgG1 3 mg/kg

LFA102 0.3 mg/kgLFA102 0.1 mg/kgLFA102 0.01 mg/kg

% A

nim

als

rem

ain

ing

Time to progression (d)

** **

0 5 10 151 × 1005

1 × 1006

1 × 1007

1 × 1008

1 × 1009

1 × 1010

IgG1 3 mg/kg

LFA102 10 mg/kgLFA102 3 mg/kgLFA102 1 mg/kg

LFA102 0.3 mg/kgLFA102 0.1 mg/kgLFA102 0.01 mg/kg

**

**

Days after randomization

Ph

oto

n e

mis

sio

n

BIgG1 Control IgG1 Control + PRL

LFA102 LFA102 + PRL

IgG1 3 mg/kg

LFA102 10 mg/kg






oPRL-induced Stat5 phosphorylation in Nb2-11-luctumors (Fig. 4A). To assess antitumor activity of LFA102,mice with established tumors received a single dose ofLFA102 (ranging from 0.01 to 10 mg/kg) or control anti-body. Disease burden was evaluated by bioluminescenceimaging through day 14 post-dosing and time to progres-sion assessed for 4.5months.A single treatment of LFA102at dose levels �0.3 mg/kg resulted in disease regressionby day 3 post-dosing, with significant efficacy relative tothe control group sustained until the final day of imaging(P < 0.05; Fig. 4B). Themean photon count on day 14 post-dosing in the IgG1 isotype control group was 1.7 � 109

photons/s whereas that of the 10 mg/kg LFA102 treatedgroupwas 5.6� 105 photons/s, approximately the lowestlevel of detection (Fig. 4C). Time to progression in thegroups receiving �0.3 mg/kg LFA102 was significantlylonger relative to the control group (P < 0.05; Fig. 4D).These results indicate that LFA102 is highly efficacious ina PRL-dependent cancer model in mice.

LFA102 PK/PD relationship and efficacy in DMBA-induced rat mammary tumor model

A paucity of biologically relevant preclinical models,perhaps driven by the lack of hPRLR–activating PRL instandard cell culture media, has contributed to the diffi-culty in assessing the relevance of PRL as an oncogenicdriver in human breast cancer (37). In addition, as mPRLdoes not appreciably activate hPRLR (Fig. 2), humanbreast cancer cells or primary tumors cannot be used asviable in vivo efficacy models in standard immunocom-promisedmice (39). Similarly,with the exception ofMCF7cells, few nonselected or nonengineered ER-positivehuman breast cancer cell lines have maintained estrogensensitivity or are suitable for use as in vivo efficacymodels.

7,12-Dimethylbenz[a]anthracene (DMBA) carcinogen–induced rat mammary tumors represent widely usedmodels of hormone-sensitive breast cancer that havebeen used previously to justify the clinical use of aroma-tase inhibitors (40, 41). A transplantable mammary tumormodel expressing both PRLR and ERa (Fig. 5A) wasderived from a primary DMBA-induced tumor and usedto conduct a PK, PD, and efficacy evaluation of LFA102 asamonotherapy. To compensate for the abbreviated serumhalf-life of LFA102 in female rats (Supplementary TableS1), multiple doses of the antibody were used to achievean effective systemic exposure. Phospho-Stat5 levels inthe tumors were analyzed by IHC 48 hours after the finalLFA102 dose in this study. A correlation between LFA102concentration in serum and inhibition of intratumoral p-Stat5 levels was observed (Fig. 5B). A significant antitu-mor effect of LFA102 treatment was also observed fromstudy days 10 through 27 post-dosing (SupplementaryFig. S6; P < 0.05), with 33% of tumors regressing withLFA102 treatment compared with 11%with vehicle treat-ment (1 case of spontaneous regression). In a secondstudy, the efficacy of LFA102 and the aromatase inhibitorletrozole was evaluated singly and in combination. Treat-ment with LFA102 alone or in combination with letrozole

SerumLFA102

Serum LFA102

PRLR ER-α

Vehicle

202 μg/mL 70 μg/mL

12 μg/mL 18 μg/mL

A

B

C

3

0 1

2 3

Days after randomization

Me

an

tu

mo

r v

olu

me

(m

m3)

± S

EM LFA102 300 mg/kg

LFA102 + Letrozole

0 5 10 15 20

*

* +

Letrozole 10 μg/kg

Vehicle2,500

2,000

1,500

1,000

500

0

Figure 5. The DMBA-induced mammary tumor model expresses PRLRand ERa and is responsive to LFA102. A, representative images of PRLRand ERa immunostaining in tumors (�400). B, the degree of inhibition ofp-Stat5 in tumors correlateswith LFA102 serum levels (data fromLFA102monotherapy study). Representative IHC images of p-Stat5immunostaining of tumors from treated rats are shown (�200) withsemiquantitative p-Stat5 scores in the top right-hand corner (3, high;2, med; 1, low; 0, negative); corresponding LFA102 serum levels areindicatedbelow images. C, LFA102 inhibits the growth of DMBA-inducedmammary tumors as a single agent and in combination with letrozole.Dosing initiation indicated by arrow. Tumor volume data are presented asmean � SEM. Statistically significant efficacy of LFA102 and thecombination treatment was shown on days 9 to 20 post-dosing(�, P < 0.05). The activity of LFA102 plus letrozole was significantly higherthan letrozole alone on days 6 to 16 post-dosing (þ, P < 0.5).

Damiano et al.





resulted in significant efficacy relative to vehicle admin-istration on days 9 to 20 post-dosing (Fig. 5C; P < 0.05).Notably, the activity of the combination treatment washigher than letrozole as a single agent on days 6 to 16 post-dosing (P < 0.05). Tumor regressions were observed inboth the LFA102 monotherapy and combination groupson day 20 (20% and 47% of tumors, respectively); how-ever, regressionswere not observed in the animals treatedwith letrozole alone or with vehicle (Table 1). Additionalstudies are being conducted to understand why someLFA102-treated tumors (as a single agent or in combina-tion) completely regressed whereas others showed only agrowth inhibition. It is unclearwhether or not even higherlevels of LFA102 efficacy could have been achieved inthese studies had antibody clearance and variability inexposure not been an issue in this species.Nobodyweightloss or other clinical observations were observed in eitherstudy. Thus, LFA102 modulates PRLR signaling activity,inhibits tumor growth, and is well tolerated in a mam-mary cancer model expressing both PRLR and ER.

DiscussionTo date, antagonists targeting the PRL/PRLR signaling

axishavehadassociated liabilities thatprecluded theirusein understanding the role of prolactin in human cancer.The clinical use of mutant PRL peptide antagonists,including G129R-hPRL, D1-9-G129R-hPRL, and S179D-hPRL, has been limited by factors such as agonism, lowpotency, and short systemic half-lives in animals (24,25, 28, 42, 43). For example, although D1-9-G129R-hPRLdoes not exhibit agonism, it requires a circulating concen-trationof 1 to 2mmol/L to effectively inhibit autocrinePRLfunction in a mouse model of prostate cancer (25). Thislevel of drug is likely to be a challenge to achieve chron-ically inhumansgiven that the reported serumT1/2 ofPRLand its peptide antagonists is approximately 20 minutes(44, 45). Anti-prolactinemic agents that act directly on thepituitary gland, such as bromocriptine, fail to impact theeffects of PRL derived from non-pituitary tissue sources(including tumor autocrine/paracrine PRL) or to inhibithGH-mediated activation of PRLR and thus fall short offully neutralizing PRLR activity systemically (19). Here,wehavedescribed the preclinical development of amono-clonal antibody that specifically binds and neutralizesPRLR with high potency. In vitro assays using PRL-sen-

sitive human breast cancer cell lines and PRL-dependentrodent cell lines have indicated that LFA102 is an effectiveantagonist of PRLR function. Furthermore, through theuse of mechanistic cell line models, we have also shownthat LFA102 is capable of neutralizing the effects of auto-crine/paracrine PRL aswell as high levels of exogenouslyadded PRL in vitro, differentiating LFA102 from moststrictly ligand-competitivePRLRantagonists.On thebasisof the non–ligand-competitive nature of LFA102 bindingand the location of its epitope in the D2 domain of PRLR,we hypothesize that LFA102 antagonizes receptor func-tion by either blocking receptordimerizationor by lockingthe dimer into an inactive conformation. Further experi-mentation is needed to better define the structure–func-tion relationship of LFA102–PRLR interactions.

LFA102 has shown an ability to effectively antagonizePRL-induced signaling in 3 tumor xenograft models andto induce the regression of PRL-dependent Nb2-11-luctumor xenografts, leading to prolonged time to progres-sion after a single administration. We have also shownthat at therapeutically relevant concentrations, LFA102has the capacity tomediate ADCC, amechanism of actionshared by many clinically successful monoclonal antibo-dies (46, 47). The observed LFA102-induced increase inserum PRL levels in rats is suggestive that systemic PRLRneutralizationhadbeenachieved in these animals and thisendpoint is being explored further as a potential biomark-er of LFA102 activity. High levels of LFA102 exposurewere found to be extremely well tolerated in preclinicalsafety studies (data not shown), consistentwith the lack ofovert toxicity observed in PRLR knockout mice (48). Thissuggests there could be few significant toxicologic con-sequences of abrogating PRLR signaling with this antag-onist in humans.

The demonstration that LFA102 was able to achievesignificant antitumor efficacy as a monotherapy, includingtumor regressions, in the DMBA-induced rat mammarycancermodelwas particularly encouraging. Given that thein vivo efficacy of letrozole may be enhanced through theaddition of LFA102, a combination therapeutic regimenconsisting of LFA102 with a standard of care (e.g., tamox-ifen or an aromatase inhibitor) could lead to greater anti-tumor efficacy in patients, compared with either agentalone. The high dose levels of LFA102 used in studies withfemale rats (to compensate for abnormal antibody PK in

Table 1. Tumor response to LFA102 and letrozole in the DMBA tumor model

Drug treatmentsMean tumorvolume (�SEM)

% Controltumor volume

% Tumorregressionsa

Vehicle 1,964 � 243 100 0LFA102 (300 mg/kg)b 809 � 279 34 20Letrozole (10 mg/kg) 1,004 � 196 45 0LFA102 þ letrozole 436 � 144 13 47

aThree consecutive measurements �50% of tumor volume at randomization.bHigh dose level used to compensate for abnormally high LFA102 clearance in female rats.






this species) are not expected to be needed to achieveeffective PRLRneutralization in humans, based onLFA102PK in male rats and primates (data not shown) and effica-cious dose levels of LFA102 in mice. We are also investi-gating whether activation of the PRL/PRLR signaling axiscould constitute amode of tumor escape from the effects ofanti-estrogens or other breast cancer therapeutics, inwhichcase LFA102 combination approaches could have use instemmingtheemergenceofdrugresistanceandprolongingthe durability of clinical responses. The therapeutic prop-erties of LFA102 described here suggest that this antibodyhas the potential to become a first-in-class anti-cancermedicine and to address unmet medical need through aunique targeted approach.A clinical trial has been initiatedto examine the safety, pharmacokinetics, and preliminaryevidence of efficacy of this antagonist in patients withmetastatic breast cancer.

Disclosure of Potential Conflicts of InterestJ.S. Damiano is employed in Novartis as Senior Research Investigator

and has ownership interest in Novartis. M. Ghoddusi has ownershipinterest. A. Fanidi has ownership interest in Novartis. T.J. Abrams andJ.A. Abraham have ownership interest in Novartis. J.S. Damiano,M. Ghoddusi, A. Fanidi, T.J. Abrams, and J.A. Abraham are authors ofNovartis patents, but have no ownership interest in the patents. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: J.S. Damiano, K.G. Rendahl, A. Fanidi, T.J.Abrams, J.A. AbrahamDevelopment ofmethodology: J.S. Damiano, K.G. Rendahl,M.Ghoddusi,J. HolashAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.S. Damiano, K.G. Rendahl, C. Karim, M.G.Embry, M. Ghoddusi, J. HolashAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): J.S. Damiano, K.G. Rendahl, C. Karim,M.G.Embry, M. Ghoddusi, J. HolashWriting, review, and/or revision of the manuscript: J.S. Damiano, K.G.Rendahl, M. Ghoddusi, J. Holash, A. Fanidi, T.J. Abrams, J.A. AbrahamAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K.G. Rendahl, C. Karim, M.G.Embry, J. HolashStudy supervision: J.S. Damiano, K.G. Rendahl, T.J. Abrams

AcknowledgmentsThe authors thank Nancy Pryer, Susmita Chatterjee, Janine Kline,

Ruben Flores, Jose Lapitan, Sok Chey, and Novartis Comparative Med-icine for their contributions to LFA102 efficacy and pharmacokineticexperiments; and Mohammad Luqman, Eric Fang, Ursula Jeffry, WeiZhang, Natasha Aziz, John Rediske, Shefali Kakar, and Xoma LLC fortheir contributions to the work presented here.

The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked adver-tisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 7, 2012; revised December 3, 2012; acceptedDecember 18, 2012; published OnlineFirst December 27, 2012.

References1. Tworoger SS, Eliassen AH, Sluss P, Hankinson SE. A prospective

study of plasma prolactin concentrations and risk of premenopausaland postmenopausal breast cancer. J Clin Oncol 2007;25:1482–8.

2. Wang PS, Walker AM, Tsuang MT, Orav EJ, Glynn RJ, Levin R, et al.Dopamine antagonists and the development of breast cancer. ArchGen Psychiatry 2002;59:1147–54.

3. Rose-Hellekant TA, Arendt LM, Schroeder MD, Gilchrist K, SandgrenEP, Schuler LA. Prolactin induces ERalpha-positive and ERalpha-negative mammary cancer in transgenic mice. Oncogene 2003;22:4664–74.

4. Arendt LM, Rugowski DE, Grafwallner-Huseth TA, Garcia-BarchinoMJ, Rui H, Schuler LA. Prolactin-induced mouse mammary carcino-mas model estrogen resistant luminal breast cancer. Breast CancerRes 2011;13:R11.

5. Touraine P, Martini JF, Zafrani B, Durand JC, Labaille F, Malet C, et al.Increased expression of prolactin receptor gene assessed by quan-titative polymerase chain reaction in human breast tumors versusnormal breast tissues. J Clin Endocrinol Metab 1998;83:667–74.

6. Xu J, Zhang Y, Berry PA, Jiang J, Lobie PE, Langenheim JF, et al.Growth hormone signaling in human T47D breast cancer cells: poten-tial role for a growth hormone receptor-prolactin receptor complex.Mol Endocrinol 2011;25:597–610.

7. Longhi SA, Blank VC, Roguin LP, Cristodero M, Retegui LA. Relativelocalization of the prolactin receptor binding sites for lactogenichormones. Growth Horm IGF Res 2001;11:324–8.

8. Acosta JJ, Munoz RM, Gonzalez L, Subtil-Rodriguez A, Dominguez-CaceresMA,Garcia-Martinez JM, et al. Srcmediates prolactin-depen-dent proliferation of T47D and MCF7 cells via the activation of focaladhesion kinase/Erk1/2 and phosphatidylinositol 3-kinase pathways.Mol Endocrinol 2003;17:2268–82.

9. Aksamitiene E, Achanta S, Kolch W, Kholodenko BN, Hoek JB,Kiyatkin A. Prolactin-stimulated activation of ERK1/2 mitogen-acti-vated protein kinases is controlled by PI3-kinase/Rac/PAK signalingpathway in breast cancer cells. Cell Signal 2011;23:1794–805.

10. Sakamoto K, Creamer BA, Triplett AA, Wagner KU. The Januskinase 2 is required for expression and nuclear accumulation of

cyclin D1 in proliferating mammary epithelial cells. Mol Endocrinol2007;21:1877–92.

11. Carver KC, Piazza TM, Schuler LA. Prolactin enhances insulin-likegrowth factor I receptor phosphorylation by decreasing its associationwith the tyrosine phosphatase SHP-2 in MCF-7 breast cancer cells.J Biol Chem 2010;285:8003–12.

12. Carver KC, Schuler LA. Prolactin does not require insulin-likegrowth factor intermediates but synergizes with insulin-like growthfactor I in human breast cancer cells. Mol Cancer Res 2008;6:634–43.

13. Huang Y, Li X, Jiang J, Frank SJ. Prolactinmodulates phosphorylation,signaling and trafficking of epidermal growth factor receptor in humanT47D breast cancer cells. Oncogene 2006;25:7565–76.

14. Ben-Jonathan N, Chen S, Dunckley JA, LaPensee C, Kansra S.Estrogen receptor-alpha mediates the epidermal growth factor-stim-ulated prolactin expression and release in lactotrophs. Endocrinology2009;150:795–802.

15. Yamauchi T, Yamauchi N, Ueki K, Sugiyama T, Waki H, Miki H, et al.Constitutive tyrosine phosphorylation of ErbB-2 via Jak2 by autocrinesecretion of prolactin in human breast cancer. J Biol Chem 2000;275:33937–44.

16. Fritze D, Queisser W, Schmid H, Kaufmann M, Massner B, Wester-hausen M, et al. Prospective randomized trial concerning hyper- andnormoprolactinemia and the use of bromoergocryptine in patientswithmetastatic breast cancer. Onkologie 1986;9:305–12.

17. Holtkamp W, Nagel GA. [Bromocriptine in chemotherapy-resistant,metastatic breast cancer. Results of the GO-MC-BROMO 2/82 AIOStudy]. Onkologie 1988;11:121–7.

18. Ben-Jonathan N, Liby K, McFarland M, Zinger M. Prolactin as anautocrine/paracrine growth factor in human cancer. Trends EndocrinolMetab 2002;13:245–50.

19. Clevenger CV, Plank TL. Prolactin as an autocrine/paracrine factor inbreast tissue. J Mammary Gland Biol Neoplasia 1997;2:59–68.

20. Hugo ER, Borcherding DC, Gersin KS, Loftus J, Ben-Jonathan N.Prolactin release by adipose explants, primary adipocytes, and LS14adipocytes. J Clin Endocrinol Metab 2008;93:4006–12.

Damiano et al.





21. Milewicz T, Rys J, Wojtowicz A, Stochmal E, Jach R, Krzysiek J, et al.Overexpression of P53 protein and local hGH, IGF-I, IGFBP-3, IGFBP-2 and PRL secretion by human breast cancer explants. Neuro Endo-crinol Lett 2011;32:328–33.

22. Wu ZS, Yang K, Wan Y, Qian PX, Perry JK, Chiesa J, et al. Tumorexpression of human growth hormone and human prolactin predict aworse survival outcome in patients with mammary or endometrialcarcinoma. J Clin Endocrinol Metab 2011;96:E1619–29.

23. Li H, Ahonen TJ, Alanen K, Xie J, LeBaron MJ, Pretlow TG, et al.Activation of signal transducer and activator of transcription 5 inhuman prostate cancer is associated with high histological grade.Cancer Res 2004;64:4774–82.

24. Bernichtein S, Kinet S, Jeay S, Llovera M, Madern D, Martial JA, et al.S179D-human PRL, a pseudophosphorylated human PRL analog, isan agonist and not an antagonist. Endocrinology 2001;142:3950–63.

25. Goffin V, Bernichtein S, Touraine P, Kelly PA. Development andpotential clinical uses of humanprolactin receptor antagonists. EndocrRev 2005;26:400–22.

26. ChenTJ,KuoCB,TsaiKF,LiuJW,ChenDY,WalkerAM.Developmentofrecombinant human prolactin receptor antagonists by molecular mim-icry of the phosphorylated hormone. Endocrinology 1998;139:609–16.

27. Chen WY, Ramamoorthy P, Chen N, Sticca R, Wagner TE. A humanprolactin antagonist, hPRL-G129R, inhibits breast cancer cell prolifer-ation through induction of apoptosis. Clin Cancer Res 1999;5:3583–93.

28. Clevenger CV, Zheng J, Jablonski EM, Galbaugh TL, Fang F. Frombench to bedside: future potential for the translation of prolactininhibitors as breast cancer therapeutics. J Mammary Gland BiolNeoplasia 2008;13:147–56.

29. Dagil R, Knudsen MJ, Olsen JG, O'Shea C, Franzmann M, Goffin V,et al. The WSXWS motif in cytokine receptors is a molecular switchinvolved in receptor activation: insight from structures of the prolactinreceptor. Structure 2012;20:270–82.

30. Clevenger CV, Furth PA, Hankinson SE, Schuler LA. The role ofprolactin in mammary carcinoma. Endocr Rev 2003;24:1–27.

31. van AJ, Zhang C, Tallet E, Raynal B, Hoos S, Baron B, et al. Structuralcharacterization of the stem-stem dimerization interface betweenprolactin receptor chains complexed with the natural hormone. J MolBiol 2010;404:112–26.

32. Baselga J, TripathyD,Mendelsohn J, BaughmanS,BenzCC,Dantis L,et al. Phase II study of weekly intravenous recombinant humanizedanti-p185HER2monoclonal antibody in patients with HER2/neu-over-expressing metastatic breast cancer. J Clin Oncol 1996;14:737–44.

33. Binart N,HellocoC,OrmandyCJ,Barra J,Clement-Lacroix P,BaranN,et al. Rescue of preimplantatory egg development and embryo implan-tation in prolactin receptor-deficient mice after progesterone admin-istration. Endocrinology 2000;141:2691–7.

34. Perry JK, Mohankumar KM, Emerald BS, Mertani HC, Lobie PE. Thecontribution of growth hormone to mammary neoplasia. J MammaryGland Biol Neoplasia 2008;13:131–45.

35. Zuckier LS,Georgescu L, ChangCJ, ScharffMD,Morrison SL. The useof severe combined immunodeficiency mice to study the metabolismof human immunoglobulin G. Cancer 1994;73 Suppl:794–9.

36. Smirnova OV, Petraschuk OM, Kelly PA. Immunocytochemical local-ization of prolactin receptors in rat liver cells: I. Dependence on sex andsex steroids. Mol Cell Endocrinol 1994;105:77–81.

37. Biswas R, Vonderhaar BK. Role of serum in the prolactin responsive-ness of MCF-7 human breast cancer cells in long-term tissue culture.Cancer Res 1987;47:3509–14.

38. Utama FE, Tran TH, Ryder A, LeBaron MJ, Parlow AF, Rui H. Insen-sitivity of human prolactin receptors to nonhuman prolactins: rele-vance for experimental modeling of prolactin receptor-expressinghuman cells. Endocrinology 2009;150:1782–90.

39. Utama FE, LeBaron MJ, Neilson LM, Sultan AS, Parlow AF, WagnerKU, et al.Humanprolactin receptors are insensitive tomouseprolactin:implications for xenotransplant modeling of human breast cancer inmice. J Endocrinol 2006;188:589–601.

40. Schieweck K, Bhatnagar AS, Batzl C, Lang M. Anti-tumor andendocrine effects of non-steroidal aromatase inhibitors on estro-gen-dependent rat mammary tumors. J Steroid Biochem Mol Biol1993;44:633–6.

41. Dukes M. The relevance of preclinical models to the treatment ofpostmenopausal breast cancer. Oncology 1997;54 Suppl 2:6–10.

42. Chen NY, Holle L, Li W, Peirce SK, Beck MT, Chen WY. In vivo studiesof the anti-tumor effects of a human prolactin antagonist, hPRL-G129R. Int J Oncol 2002;20:813–8.

43. Schroeder MD, Brockman JL, Walker AM, Schuler LA. Inhibition ofprolactin (PRL)-induced proliferative signals in breast cancer cells by amolecular mimic of phosphorylated PRL, S179D-PRL. Endocrinology2003;144:5300–7.

44. Clark R, Olson K, Fuh G, Marian M, Mortensen D, Teshima G, et al.Long-acting growth hormones produced by conjugation with poly-ethylene glycol. J Biol Chem 1996;271:21969–77.

45. Rouet V, Bogorad RL, Kayser C, Kessal K, Genestie C, Bardier A, et al.Local prolactin is a target to prevent expansion of basal/stem cells inprostate tumors. Proc Natl Acad Sci U S A 2010;107:15199–204.

46. Varchetta S, Gibelli N, Oliviero B, Nardini E, Gennari R, Gatti G,et al. Elements related to heterogeneity of antibody-dependent cellcytotoxicity in patients under trastuzumab therapy for primaryoperable breast cancer overexpressing Her2. Cancer Res 2007;67:11991–9.

47. Bibeau F, Lopez-Crapez E, Di FF, Thezenas S, Ychou M, Blanchard F,et al. Impact of Fc{gamma}RIIa-Fc{gamma}RIIIa polymorphisms andKRAS mutations on the clinical outcome of patients with metastaticcolorectal cancer treated with cetuximab plus irinotecan. J Clin Oncol2009;27:1122–9.

48. Ormandy CJ, Camus A, Barra J, Damotte D, Lucas B, Buteau H, et al.Null mutation of the prolactin receptor gene produces multiple repro-ductive defects in the mouse. Genes Dev 1997;11:167–78.






2013;12:295-305. Published OnlineFirst December 27, 2012.Mol Cancer Ther Jason S. Damiano, Katherine G. Rendahl, Christopher Karim, et al. of Breast CancerAntibody LFA102, a Novel Potential Therapeutic for the Treatment Neutralization of Prolactin Receptor Function by Monoclonal

Updated version

10.1158/1535-7163.MCT-12-0886doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2013/01/02/1535-7163.MCT-12-0886.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/12/3/295.full#ref-list-1

This article cites 48 articles, 14 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/12/3/295.full#related-urls

This article has been cited by 2 HighWire-hosted articles. Access the articles 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/12/3/295To 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-12-0886

http://mct.aacrjournals.org/content/suppl/2013/01/02/1535-7163.MCT-12-0886.DC1

http://mct.aacrjournals.org/content/12/3/295.full#ref-list-1

http://mct.aacrjournals.org/content/12/3/295.full#related-urls

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

mailto:[email protected]

http://mct.aacrjournals.org/content/12/3/295


neutralizationofprolactinreceptorfunctionbymonoclonal...

Documents