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Published OnlineFirst September 9, 2010; DOI: 10.1158/1078-0432.CCR-10-1343

Clinical
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cer Therapy: Preclinical

03732010: A Fully Human Monoclonal Antibody against

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adherin with Antitumor and Antimetastatic Activity

C. Zhang1, Zhengming Yan1, Qin Zhang1, Kyle Kuszpit4, Kenneth Zasadny4, Ming Qiu1, Cory L. Painter1,
ny Wong2, Eugenia Kraynov3, Maria E. Arango1, Pramod P. Mehta1, Ian Popoff1, Gerald F. Casperson5, Los1, Steve Bender1, Kenna Anderes1, James G. Christensen1, and Todd VanArsdale1
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pose: P-cadherin is a membrane glycoprotein that functionally mediates tumor cell adhesion,eration, and invasiveness. We characterized the biological properties of PF-03732010, a humanclonal antibody against P-cadherin, in cell-based assays and tumor models.erimental Design: The affinity, selectivity, and cellular inhibitory activity of PF-03732010 werein vitro. Multiple orthotopic and metastatic tumor models were used for assessing the antitumorntimetastatic activities of PF-03732010. Treatment-associated pharmacodynamic changes were alsoigated.ults: PF-03732010 selectively inhibits P-cadherin–mediated cell adhesion and aggregation in vitro.P-cadherin–overexpressing tumor models, including MDA-MB-231-CDH3, 4T1-CDH3, MDA-MB-AL-CDH3, HCT116, H1650, PC3M-CDH3, and DU145, PF-03732010 inhibited the growth ofry tumors and metastatic progression, as determined by bioluminescence imaging. Computedraphy imaging, H&E stain, and quantitative PCR analysis confirmed the antimetastatic activity of732010. In contrast, PF-03732010 did not show antitumor and antimetastatic efficacy in the coun-t tumor models exhibiting low P-cadherin expression. Mechanistic studies via immunofluorescence,nohistochemical analyses, and 3′-[18F]fluoro-3′-deoxythymidine–positron emission tomographyng revealed that PF-03732010 suppressed P-cadherin levels, caused degradation of membranenin, and concurrently suppressed cytoplasmic vimentin, resulting in diminished metastatic capac-hanges in the levels of Ki67, caspase-3, and 3′-[18F]fluoro-3′-deoxythymidine tracer uptake alsoted antiproliferative activity and increased apoptosis in the tested xenografts.clusions: These findings suggest that interrupting the P-cadherin signaling pathway may be a novel
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therapeutic approach for cancer therapy. PF-03732010 is presently undergoing evaluation in Phase 1 clin-ical trials. Clin Cancer Res; 16(21); 5177–88. ©2010 AACR.

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classic cadherins constitute a superfamily of mole-that mediate calcium-dependent cell-cell adhesions.tracellular domains of cadherins directly bind to β-n and link with cytoskeletal components, providing

basis for stable cell-cell adhesion (1). Thein signaling pathway also represents a

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in areproteimostfunctisupprmerouloss otion oimpliccadhelial-msioncadhe

s: 1Oncology Research Unit, 2Drug Safety Researchand 3Pharmacokinetics, Dynamics and Metabolism,arch and Development, La Jolla Laboratories, SanBio-Imaging Unit, Groton Laboratories, Pfizer Globalvelopment, Groton, Connecticut; and 5ProteinPfizer Global Research and Development, St. Louisouis, Missouri

ry data for this article are available at Clinical Cancerttp://clincancerres.aacrjournals.org/).

thor: Cathy Zhang, Translational Research Group inh Unit, Pfizer Global Research and Development,nter Rd., San Diego, CA 92121. Phone: 858-622--5999; E-mail: [email protected].

0432.CCR-10-1343

ssociation for Cancer Research.

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Research. on August 31, 2clincancerres.aacrjournals.org d from

r regulatory mechanism for oncogenic signalingays (2, 3) that guide cell fate decisions throughgulation of cell growth, differentiation, motility,urvival (4). Several cadherins, such as E-cadherin,herin, VE-cadherin, P-cadherin, and cadherin 11,een implicated in cancer disease progression (5).ong the cadherin families, E-cadherin and N-cadher-the most highly characterized subgroup of adhesionns. E-cadherin is ubiquitously expressed throughoutepithelial tissues and serves as a negative regulator toonally block the β-catenin signaling pathway andess tumor cell growth (6, 7) and invasion (8, 9). Nu-s preclinical and clinical studies have shown that thef E-cadherin occurs concurrently with the upregula-f N-cadherin (10) or other cadherin family membersated in invasive growth (5). This process, known asrin switching, has been reported to promote epithe-esenchymal transition and leads to tumor cell inva-
and metastasis. N-cadherin overexpression viarin switching was observed in various invasive cancer
5177

021. © 2010 American Association for Cancer

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Translational Relevance

P-cadherin–mediated adherens junctions and the as-sociated signaling pathway play diverse roles in theregulation of tumor cell proliferation, invasiveness,and metastatic potential. Upregulation of P-cadherinwas frequently observed in various malignancies, in-cluding breast, colon, lung, and pancreatic tumors,and P-cadherin increase correlated with poor survivalof breast cancer patients. PF-03732010, a novel, highlyselective human monoclonal antibody against P-cadherin, showed antitumor and antimetastatic activityin a diverse panel of P-cadherin–overexpressing tumormodels without introducing any adverse effects. Mech-anistic studies revealed that PF-03732010 disrupted theP-cadherin signaling pathway, suppressed β-catenintranscriptional targets, and resulted in an antimetastaticand antiproliferative activity and the induction ofapoptosis. Thus, antagonizing P-cadherin withPF-03732010 represents a novel approach for antican-cer therapy targeting tumors with high P-cadherinexpression.

Zhang et al.

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es and tumors and, therefore, is emerging as a poten-erapeutic target. ADH-1, a peptide antagonist thatts N-cadherin–mediated adhesion, has been shownibit cell growth and motility in vitro, as well as tumorh and invasion in vivo (11, 12); ADH1 is currently un-ing phase 1 clinical trials.dherin (CDH3), which shares moderate homology-cadherin (41%) and E-cadherin (46%) in the extra-r domain (13), was often reported to correlate withsed tumor cell motility and invasiveness when over-ssed (14–16). P-cadherin–associated tumor cellh, motility, and invasiveness is likely or at least par-mediated by the relocalization of membrane β-cate-the cytoplasm and subsequently into the nucleusit partners with the T-cell factor and activates tran-on of the target genes (14, 17), including vimentincyclin D1, survivin, and BCL-2 (19). The upregula-f P-cadherin was frequently observed in various ma-nt tumors, including breast, colon, lung, andeatic tumors, and correlated with poor survival ofcancer patients (15, 20, 21). In contrast, significant-levels of the P-cadherin gene expression were de-in a diverse panel of normal tissues (22). Thus,tion of P-cadherin signaling represents an intriguingtunity for the development of novel targeted thera-agents.

03732010, a humanized monoclonal antibody, wasered to antagonize P-cadherin–regulated cell-cellion and the associated signaling pathway. Becauseerin is implicated in tumor cell invasion, prolifera-

and metastatic progression, in this report, we usedaneous and experimental metastasis models to eval-

presseitored

ancer Res; 16(21) November 1, 2010

Research. on August 31, 2clincancerres.aacrjournals.org ownloaded from

the antitumor and antimetastatic activity of PF-010 as well as the underlying mechanism. Noninva-ioluminescence imaging (BLI) technology was usedgitudinallymonitor disease progression.We also usedF]fluoro-3′-deoxythymidine ([18F]FLT)–positronion tomography imaging (PET) to assess the anti-erative activity of PF-03732010 and determine thetial clinical utility of this imaging modality as aate biomarker for proof of mechanism (23). Thesegs provide insights into the P-cadherin signalingay and warrant the clinical investigation of PF-010 as a novel agent for anticancer therapy.

rials and Methods

PF-03732010 humanized monoclonal antibodyenerated by affinity purification at Pfizer St. Louisn Therapeutics group (Pfizer, Inc.). D-Luciferin wasased from Caliper Life Sciences. The antibodiesinclude anti-β-catenin (BioSource International),i67 (Lab Vision), and anti-vimentin (Dako). Anti-herin, anti-E-cadherin, anti-N-cadherin, anti-D1, anti-caspase-3, anti-survivin, and anti-Bcl-2

purchased from Cell Signaling. Unless otherwise, all cell lines and fine chemicals were purchasedAmerican Type Culture Collection and Sigma-h, respectively.

ro selectivity over E-cadherindherin antibody–binding assessment was tested viascence-activated cell sorting analysis. A549 cells wereed with serum and incubated with either 50 μg/mL732010 or an E-cadherin–specific antibody (R&Ds) at 5 μg/mL for 1 hour. Cells were subsequently

ssed according to the manufacturer's instructions anded using a FACSCalibur system (BD Biosciences).

fection of tumor cell lines145, H1650, and HCT116 cell lines, which expresslevels of endogenous P-cadherin, were transfectedluciferase expression vector as previously reportedThe paired isogenic cell lines, MDA-MB-231-CDH3herin)/MDA-MB-231-pCL (vector), PC3M-CDH3/-pCL (from PC3M-luc-C6, Xenogen-Caliper Compa-nd MDA-MB-435HAL-CDH3/MDA-MB-435HAL-25, 26), were derived so that they differ only in P-rin expression. Full-length human or mouse P-cad-cDNAs were cloned into the retroviral transfer plas-CLNCX (Imgenex), and retroviral vectors werered in the PT67 packaging cell line (Clontech), ac-g to the manufacturer's protocols. 4T1-CDH3 cellstransfected to express high levels of P-cadherin. Tar-ll lines were transduced with retroviral vectors, andesistant pools of cells were selected with geneticin atg/mL. DU145, H1650, and HCT116, along with the-MB-435HAL and PC3M isogenic pairs, stably ex-
d luciferase to allow disease progression to be mon-by BLI.
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ggregation and spheroid disruption assaysperform the aggregation assay, cells were detachedsuspended as single cells at a concentration of 1 ×lls/mL. Cells were plated into 96-well poly-HEMA–plates and incubated with and without the test

s) at 37°C on a horizontal shaker for 16 hours toate aggregate formation. For the spheroid disruptioniments, cell aggregates were formed before being, and activity was expressed as fold increase in dis-cells compared with untreated controls.

ls or aggregates were fixed with 4% formaldehyde,e degree of aggregation was scanned and calculat-ng a Cellomics ArrayScan (Thermo Fisher Scientif-uipped with Target Acquisition software. Maximalition was defined as the value when EGTA wast, during which the maximum number of singlegregated cells was produced, whereas the negativel was the value when the smallest number ofll aggregates was formed in complete medium.values were determined using Graphpad Prismre.

o studies and drug administrationanimal experimental procedures complied with thefor the Care and Use of Laboratory Animals (Insti-r Laboratory Animal Research, 1996) and were ap-d by the Pfizer Global Research and Developmenttional Animal Care and Use Committee. For phar-inetic analysis, serum samples were collected untilfor ELISA analysis. PF-03732010 exhibited a half-lifeto 6.6 days in mice via s.c. or i.v. dosing (Supple-ry Fig. S1). The once-weekly s.c. dosing scheduleso used in the subsequent in vivo experiments for ef-and pharmacodynamic end point evaluations. Forficacy assessments, mice (Charles River) were ran-y assigned to groups (12 mice per group) beforeent, such that the mean tumor size was equal foroups. In studies assessing efficacy, PF-03732010dministered s.c. once per week for 6 to 9 weeks.l studies in this report, no adverse effects were ob-in mice treated with PF-03732010.

r modelsere combined immunodeficient-beige mice werefor all studies in this report, except when usingDH3 cells, for which normal BALB/c mice wereFor the mammary orthotopic implant, 2 × 106 cellscombined with Matrigel and injected into the micethe mammary fat pad. Details for the prostate

topic and subrenal capsule (SRC) tumor modelsescribed previously (27, 28). For the H1650 ortho-or lung metastasis models, 2 × 106 cells were i.v.d through the tail vein of anesthetized mice. Tu-in the lungs were identified via bioluminescencen 2 weeks after tumor inoculation. For the16-luc liver metastasis model, mice were intrasple-
injected with 2 × 103 cells, and liver metastases
visualized within a week after tumor cell implant.est, IDweigh

acrjournals.org


te orthotopic surgical implants were done by in-g 5 × 105 tumor cells into male severe combinednodeficient mice. In the SRC model, lung metasta-re visualized at 21 to 28 days after tumor implant.er stages of the SRC and experimental metastasise models, mice exhibited physiologic signs consis-ith advanced metastatic disease, moribund miceeuthanized, and the survival times were recordedplan-Meier survival analyses.

ivo BLI was conducted using the IVIS100 system withImage acquisition and analysis software (Caliper

ciences). Anesthetized mice were i.p. injected with/kg D-luciferin and imaged 10 minutes after luciferinon.

titative PCR and Western blot analysiscirculating tumor cells (CTC) in mouse wholewere evaluated by real-time quantitative PCR) analysis of human Alu DNA sequences, whichen previously validated by laser scanning cytometryWestern blot analysis was done according to thedy manufacturer's instructions. Tumors were snap-and pulverized in a liquid nitrogen-cooled mortaranalysis.

nofluorescence and immunohistochemicalngin vitro immunofluorescence staining, cells wereon coverslips in triplicate before treatment. Whencells were fixed in 4% formaldehyde and permea-

d in 100% methanol before staining. Tumor sam-ere collected after 4 to 5 weeks of treatment andfrozen in OCT medium (VWR) or prepared intolin-fixed, paraffin-embedded tissue blocks. Theng procedure was done according to the manufac-s instructions. Fluorescence images were captureda Nikon Eclipse TE2000 fluorescent microscopeQ-Capture software, and image analysis was doneImage Pro Plus 5.1 (Media Cybernetics). Quantita-nalysis of immunohistochemical staining was donethe Chromovision automated cell imaging system.

LT-PET imaging]FLT-PET and computed tomography (CT) imagingone with a microPET Focus F220 scanner (Siemensal Solutions) and a GE eXplore microCT scannerealthcare), respectively, as described previously

The standardized uptake value (SUV) was calculatedthe following formula:

SUV ¼ CPETðTÞ=ðID=WÞ;

CPET is the measured activity in the volume of inter-
is the injected dose (μCi), andW is the mouse bodyt.
Clin Cancer Res; 16(21) November 1, 2010 5179



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analysisor growth inhibition (TGI) was calculated as 100 ×T/ΔC). The ΔC (ΔT) was obtained by subtractingean tumor volume in the vehicle (treated) groupfirst day of treatment from the mean tumor volumeday of assessment. Statistical analyses were con-using GraphPad Prism with one-way ANOVA anal-

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ty and specificity of PF-03732010 againstherinsingle-chain Fc versions of PF-03732010 bind touman and mouse P-cadherin/Fc proteins, with dis-ion constant values (Kd) of 2.5 and 1.8 nmol/L, re-ively, suggesting similar affinities toward bothns. Because the extracellular domain of human ande mature P-cadherin proteins share 87% homologyPF-03732010 showed comparable cross reactivityhuman and mouse P-cadherins. The selectivity of732010 was tested on immobilized P-cadherin ver-her related cadherins via an ELISA format (Supple-ary Fig. S2), and PF-03732010 did not exhibiticant binding to E-cadherin, N-cadherin, or VE-
rin recombinant proteins across a broad range of bition


confirm that PF-03732010 does not bind to E-rin, cell-based flow cytometric analysis was doneA549 cells, which exhibit high E-cadherin expres-ut no detectable P-cadherin expression. Figure 1Athat binding of PF-03732010 to A549 cells was

etected.

ro functional assay of PF-03732010dherin expression in the cell lines used in this reportested by immunoblot analysis (Supplementary3). High levels of P-cadherin were observed in16, DU145, and H1650, as well as the cell lineseered to express P-cadherin (CDH3). In contrast,-transfected (pCL) lines showed minimal expressionadherin.cell-based functional assays, PF-03732010 signifi-disrupted the MDA-MB-435HAL-CDH3 cell aggre-with an IC50 of 5 nmol/L (Fig. 1B and C).732010 also disrupted existing P-cadherin–mediat-hesion. When the preestablished DU145 spheroidstreated for 24 hours, PF-03732010 (>5 nmol/L) ex-d an 11-fold to 15-fold increase in disruptedoids when compared with vehicle-treated cellsC, bottom). DU145 cells treated with PF-03732010mol/L) for 24 hours showed a reduction in mem--localized β-catenin (Fig. 1D).ether, PF-03732010 showed a similar degree of inhi-
of cellular aggregation, disruption of spheroids, and
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In vitro characteristics of32010. A, PF-03732010ot show nonspecificto E-cadherin. B and C,32010 inhibits MDA-MB-L-CDH3 cell aggregateon. C, PF-03732010s DU145 spheroids.3732010 modulatesane β-catenin level incells. Images are at 40×

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adherin–expressing cell lines, including HCT116,5, MDA-MB-231-CDH3, PC3M-CDH3, and MDA-5HAL-CDH3.

ntitumor and antimetastatic properties of PF-010 against multiple tumor models, includingary, non–small cell lung, colon, and prostate

r modelsprimary tumor sizes in the 4T1-CDH3, MDA-MB-DH3, and MDA-MB-231-pCL mammary orthotopicls were assessed by caliper measurement. For othermodels, luciferase-expressing DU145, PC3M-pCL,

vehicle group. Tumor sizes in the MDA-MB-231-pCL, MDA-MB-231-CDH; tumors or metastases in other models were quantified with BLI.

-CDH3, H1650, and HCT116 lines were used to en-LI assessment of the primary tumor and metastasis.

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tested models, treatment of PF-03732010 (s.c. injec-was done once per week until the study ended.representative BLI images of selected tumor modelsA) and the quantitative measurements of the tumorns in all tested models (Fig. 2B) showed the antitu-nd antimetastatic efficacy of PF-03732010 (20 mg/a panel of orthotopic and metastasis models.

T116 liver metastasis and DU145 lung metastasisls, PF-03732010 displayed significant (P < 0.05)etastatic activity. PF-03732010 also exhibited signif-(P < 0.05) antitumor efficacy against tumor growth1-CDH3, MDA-MB-231-CDH3, DU145, PC3M-

4T1-CDH3 mammary orthotopic models were assessed with

PF-03732010 exhibits antitumor and antimetastatic properties in multiple P-cadherin–expressing tumor models. Representative BLI images ofd tumor models (A) and the quantitative assessment of the relative tumor sizes in all tested tumor models (B). In each orthotopic model, treatmenttiated when primary tumors were established. In the HCT116 and DU145 metastasis models, PF-03732010 was administered prophylactically atfore tumor cell inoculation, and the TGI was determined during the late stage of the exponential growth period in vehicle-treated mice. *, P < 0.05

, and H1650 orthotopic models. Dose-dependenty was shown in the PC3M-CDH3 orthotopic model.




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732010 at 5 and 20 mg/kg (data not shown) in-49% and 73% TGI (P < 0.05), respectively. In con-n the MDA-MB-231-pCL and PC3M-pCL orthotopicls, which expressed low levels of P-cadherin, PF-010 (20 mg/kg) did not exhibit antitumor activity.se 4T1-CDH3, MDA-MB-231-CDH3, DU145, PC3-, H1650, and HCT116 cell lines express high levelsadherin, these results suggest that the efficacy of PF-010 was dependent on the blockade of P-cadherining.he PC3M-CDH3 tumor-bearing mice, qPCR analysishuman Alu gene in mouse whole blood (at day 34)d that PF-03732010 at 5 and 20 mg/kg significantly
d the number of human CTCs compared with vehi-atment (data not shown).
reducgroup

CR analysis of human CTCs (C) on day 50. Line plots show the dose-dependen), tumor burden in lungs (E), and Kaplan-Meier survival analysis using moribund



ntitumor and antimetastatic properties of732010 in MDA-MB-435HAL-CDH3 SRC andimental metastasis tumor modelsnext tested the antitumor and antimetastatic proper-f PF-03732010 in SRC tumor models using antigen-ssing MDA-MB-435HAL-CDH3 cells, which formtaneous metastases in lungs. Treatment of PF-010 was initiated on day 9 after tumor cell implan-, and representative BLI images of the primary anddary tumor burdens on day 50 (Fig. 3A) showedntitumor and antimetastatic properties of PF-010. H&E staining of lungs from a separate cohorte mice in each group (Fig. 3B) indicated a marked
tion of the tumor burden in PF-03732010–treateds compared with vehicle controls, in agreement with
PF-03732010 displays antitumor and antimetastatic properties and survival benefit in the MDA-MB-435HAL-CDH3 SRC model. Values representan ± SEM from 12 mice. *, P < 0.05 versus vehicle group. The representative BLI images (A), H&E staining of tumor cell infiltration into lungs (B),

t inhibition of PF-03732010 against primary tumor growth instatus as the end point.




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I images. In addition, the reduction in the humanne, detected by qPCR analysis, suggested a reducedf CTCs in mice treated with PF-03732010 (Fig. 3C).ificant inhibition (P < 0.05) of the primary tumorn was observed at each dose level (Fig. 3D) evaluat-or beyond day 36 after tumor implantation. On day-03732010 at 10, 20, and 40 mg/kg showed TGI va-f 52%, 66%, and 68%, respectively, against the pri-tumor growth. In addition, PF-03732010 showedicant dose-dependent inhibition of tumor growthondary organ sites starting on day 28, with TGI va-n day 50 of 54%, 73%, and 82% for the 10, 20, and/kg–treated groups, respectively (Fig. 3E). The medi-vival time for mice treated with PF-03732010 at 10,d 40 mg/kg significantly (P < 0.05) increased to 92,nd 96 days, respectively, compared with 71 days forhicle-treated mice.further understand how the kinetics of metastasisitigated, we tested PF-03732010 in the MDA-MB-AL-CDH3 experimental lung metastasis model by di-injecting cells into the vasculature, which omits theeps of the metastatic cascade and enables the assess-of tumor cell colonization in host organs. PF-010 was administered either 24 hours before tumorjection using the prophylactic model or after tumorsstablished in lungs (at day 14) in the established tu-odel.re 4A depicts the effect of PF-03732010 on tumorn in the prophylactic setting. PF-03732010 at thed 20 mg/kg dose levels significantly (P < 0.01) inhib-mor cell colonization (80% and 90% inhibition, re-vely) compared with the vehicle-treated group, andedian survival time significantly (P < 0.01) improvedand 93 days, respectively, over 54 days for the vehi-ated mice (Fig. 4B). In the therapeutic setting, PF-2010 significantly inhibited tumor growth (P <in lungs at both 10 and 20 mg/kg dose levels withalues of 77.2% and 86.8%, respectively (Fig. 4C).evaluated dose levels also improved (P < 0.05) then survival time to 74 days, compared with 55 dayshicle-treated mice.overall results in the experimental metastasis modelthat PF-03732010 not only inhibits tumor cell col-tion in lungs but also inhibits tumor growth after be-tablished in the host organ.

732010 negatively modulates P-cadherin andnin levels, results in antiproliferation andetastatic activities, as well as increasedosisdherin mediates adherens junctions through directg of β-catenin with cadherin cytoplasmic domains.ition to serving a functional role through P-cadherinbilize cell-cell adhesion, β-catenin acts as a signal-olecule to mediate cell invasion, migration, prolif-n and survival through the transcription of target
, including vimentin, cyclin D1, survivin, and.
vimenas sh

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the efficacious doses (10, 20, and 40 mg/kg), PF-010 suppressed the levels of P-cadherin, β-catenin,

progression and the treatment effect. The IgG was also tested inrapeutic setting (C). The difference in BLI measurements of thegroups between B and C was due to interexperimental variability.

PF-03732010 inhibits tumor cell colonization and lung tumorgrowth in the MDA-MB-435HAL-CDH3 experimental metastasisValues represent themean ± SEM from 12mice. In the prophylacticthe representative BLI images (A) and line plots (B) depict the

tin, and Ki67 and increased caspase-3 activation,own in representative immunofluorescence or




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nohistochemical images (Fig. 5A) and semiquanti-analysis (Fig. 5B and C) of the biomarkers in theMB-435HAL-CDH3 SRC tumor model. These datathat the disruption of the P-cadherin/β-catenin sig-pathway corresponds with diminished tumor cell

veness, proliferation, and increased apoptosis. E-rin levels were below the level of detection in thisl; therefore, we were unable to determine whethererin plays a role in this signaling cascade.
he PC3M-CDH3 prostate orthotopic model (Fig. protei
n (A), and E-cadherin (D) are at 40× magnification. The remaining immunohistochene after mice were treated with vehicle or PF-03732010 for 5 wk.



d that the degradation of β-catenin induced by PF-010 (20 mg/kg) was accompanied by a decrease inD1 levels and the increase in caspase-3 activation,ting that tumor cell antiproliferation and apoptosisassociated with the blockade of β-catenin signaling.ession of vimentin levels was also observed afterent (not shown). PF-03732010 concurrently modu-the levels of survivin and BCL2, suggesting that apo-may result from suppression of these antiapoptotic
ns. Diminished P-cadherin levels were also observed
Western blot and immunohistochemical analyses after the treatment with PF-03732010, whereas E-cadherin

PF-03732010 suppresses P-cadherin/β-catenin signaling and reduces levels of β-catenin signaling-mediated target proteins, including vimentin,1, survivin, and Bcl2. Values represent the mean ± SEM from six mice. The representative images of tumors treated with vehicle or 20 mg/kg32010 (A) and semiquantitative analyses (B, C) of P-cadherin, β-catenin, vimentin, Ki67, and caspase-3 staining at all doses in the MDA-MB--CDH3 SRC tumors. Western blot analysis of P-cadherin, cyclin D1, survivin, and Bcl2 levels and the representative immunohistochemicalof β-catenin, E-cadherin, and caspase-3 staining (D) in PC3M-CDH3 tumors. Images of P-cadherin (A), β-catenin (A, D), P-cadherin/β-catenin (A),

mical images (A, D) are at 10× magnification. All sample analyses




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ssion remained unchanged. PF-03732010 causedr results in the DU145 (Supplementary Fig. S4)1650 orthotopic models.

732010 shows antiproliferative activity via [18F]ET imaging in the MDA-MB-435HAL-CDH3 SRCr modelDA-MB-435HAL-CDH3 SRC tumors, PF-03732010and 20 mg/kg significantly (P < 0.01) reduced thee of [18F]FLT by 26.8% and 23.4%, respectively, inry tumors on day 42 (Fig. 6A and B). The observedtion in [18F]FLT uptake also occurred in the lungr burden (Fig. 6C and D). PF-03732010 at 10 and/kg suppressed [18F]FLT uptake in the lung tumor

uptake in the primary tumor (SRC). The representative images (C) and qmor burdens. The imaging analysis was done after mice were treated with

n by 31.7% and 37.8% (P < 0.01), respectively, com-with the vehicle treatment, showing the antiproli-

antagnovel

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e activity of PF-03732010 as a downstream effect

tive analyses (D) of [ F]FLT uptake and the CT intensity in thee or PF-03732010 for 5 wk.

ed on day 55.

ssion

e, we report the preclinical characteristics of PF-2010, a human monoclonal antibody againstherin. PF-03732010 efficiently blocks P-cadherin–ted cell-cell adhesion by antagonizing the β-catenining pathway, resulting in significant antiproliferativentimetastatic activity. Because the IgG shows no no-le signs of adverse effects at the efficacious doses,

[18F]FLT-PET/CT imaging shows the antiproliferative effect of PF-03732010 in the MDA-MB-435HAL-CDH3 SRC model. Values represent theSEM from eight mice. *, P < 0.05; **, P < 0.01, between the vehicle and treated groups. The representative images (A) and quantitative analyses (B) of

18

onizing P-cadherin with PF-03732010 represents aapproach to anticancer therapy.




PF-P-cadcloselin, N-using435Hin bushowthe Igactivi03732E-cadhanalyto P-csomeally suspecifwouldPF-

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03732010 showed high specificity and selectivity forherin targeting. The IgG failed to bind to the mosty target-related family members, including E-cadher-cadherin, and VE-cadherin, as assessed by ELISArecombinant proteins. In PC3M-pCL and MDA-MB-AL-pCLmodels, which express high level of N-cadher-t lack P-cadherin, treatment with PF-03732010ed no therapeutic benefit. These data suggest thatG-associated efficacy was not due to the off-targetty by antagonizing N-cadherin. Specifically, PF-010 also lacked the ability to bind to endogenouserin in cell-based fluorescence-activated cell sorting

ses, confirming the high selectivity of the antibodyadherin over E-cadherin. Because E-cadherin shareshomology with P-cadherin and E-cadherin function-ppresses tumorigenesis or metastasis (32), the highicity of PF-03732010 for P-cadherin over E-cadherinallow a better therapeutic window to be achieved.

03732010 impairs P-cadherin–mediated cell func-nd at nanomolar concentrations, the antibody alsoed P-cadherin–mediated cell-cell adhesion and dis-preestablished spheroids.

umor models, PF-03732010 induced significant TGI,gh this antiproliferative activity was not observedcells were treated with PF-03732010 in vitro. Thispancy suggests that fully functioning P-cadherin sig-may require the cell-cell and cell-stroma crosstalk inact tumor architecture during tumorigenesis and me-is, a process that may not be recapitulated undero conditions. In the experimental system, we usedherin–deficient MDA-MB-231, 4T1, PC3M, andMB-435HAL cells to construct cell lines that stablypress P-cadherin. PF-03732010 showed significantmor activity in the 4T1-CDH3 orthotopic, MDA-1-CDH3 orthotopic, PC3M-CDH3 orthotopic, andDA-MB-435HAL-CDH3 SRC models, as well as in0 and DU145 orthotopic models, in which tumorndogenously expressed high levels of P-cadherin.mparison, tumor models with minimal P-cadherinsion, including the MDA-MB-231-CDH3 orthoto-C3M-pCL orthotopic, MDA-MB-435HAL-pCL SRClementary Fig. S5A and B), and the MDA-MB-AL-pCL experimental metastasis (Supplementary5C and D) models, showed no antitumor response03732010; these results suggest that PF-03732010nflicts efficacy in tumors with high P-cadherin ex-on. P-cadherin is frequently upregulated in a diverseof malignancies (15, 33, 34) and often associatedigh histologic grade, invasiveness, and poor survival4, 35). The broad spectrum activity of PF-03732010es rationale and opportunity in the clinical develop-of the antibody for treating patients with tumorsigh P-cadherin expression.easing evidence indicates that high P-cadherin ex-on in human cancer cells correlates with increasedotility (15) and invasiveness (20), which led us to
igate whether PF-03732010 slows down metastasisocking the P-cadherin signaling pathway. During
targetepith



etastatic cascade, malignant cells break away fromimary tumor, shed into the bloodstream or migrategh the lymphatic system, and eventually colonize ateferred organ microenvironment, as proposed in Pa-seed and soil” theory (36). PF-03732010 reducednode metastases via BLI in the PC3M-CDH3 and

5 prostate orthotopic models and lowered CTC le-n mouse whole blood of PC3M-CDH3 tumor-g mice. The antimetastatic property of the antibodyurther confirmed in the MDA-MB-435HAL-CDH3odel; via BLI and CT imaging, as well as H&E stain-lungs, PF-03732010 significantly inhibited tumorfiltration into the lungs at all doses tested (10, 20,0 mg/kg). Also using this model, PF-03732010 re-CTC levels in whole blood. Supported by the in vitroresults (data not shown), the ability of PF-03732010press CTCs in mouse whole blood could be partiallyby antibody-dependent cell-mediated cytotoxicity.

ong the millions of cells that primary tumors releasee circulation, only a small minority of them will ad-t a distant organ and eventually establish metastasesWe therefore assessed whether the antimetastaticteristic of PF-03732010 was entirely due to the re-on of CTCs in whole blood. In the MDA-MB-AL-CDH3 prophylactic metastasis model, mice wereated with PF-03732010 to ensure that plasma levelsIgG were maintained above the effective biologicalntration when the tumor cells were i.v. injected (24posttreatment). PF-03732010 significantly inhibitedr cell adhesion in the lungs, and similar results wereed in the HCT-116 and DU145 experimental metas-odels. Notably, under the same settings, MDA-MB-

AL-pCL cells did not respond to PF-03732010 in theylactic metastasis model, suggesting that the block-f MDA-MB-435HAL-CH3 colonization in the lungsue to impaired P-cadherin signaling. Overall, theetastatic activity of PF-03732010 is attributed to itsto reduce tumor cells in the circulatory system andte tumor cell colonization in the host organ.dherin knockdown in cells resulted in reduced tu-ell invasiveness regardless of E-cadherin expression(17). Indeed, loss of P-cadherin expression after732010 treatment resulted in decreased metastasisut any affect on the E-cadherin level, presumably,t, due to the reduced tumor cell adhesiveness. Moretantly, the antimetastatic activity was derived fromminished level of β-catenin, which not only playsctional role in stabilizing P-cadherin–mediatedll adhesion but also regulates molecular signalingays to promote tumor growth, invasiveness, and me-is (17, 19). It has been shown that P-cadherin faileduce invasion when the catenin-binding domain wased (38). When released from the plasma membrane,nin is stabilized by Wnt signaling and subsequentlyocates into the nucleus, where it acts as a coactivator-cell factor and induces transcriptional activation of
genes (32, 39). This pathway activation is critical forelial-mesenchymal transition, a mechanism that



contrichymasignaltumortestedPC3Mdosesβ-catestaticruptioefficachancethe s03732the Igbe fulis relenalingβ-catesubseenzymβ-ca

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butes to metastasis (40). During epithelial-mesen-l transition, one of the activated targets of β-catenining is vimentin (18), which has been implicated incell invasiveness and migratory potential. In severalxenografts, including MDA-MB-435HAL-CH3 SRC,-CDH3. and DU145 orthotopic models, efficaciousof PF-03732010 concurrently suppressed membranenin and vimentin levels and resulted in antimeta-activity, further confirming that the IgG-induced dis-n of β-catenin signaling contributes to antibodyy. In addition, as Bcl-2 overexpression leads to en-d metastatic potential in human cancers (41, 42),uppression of Bcl-2 could account for the PF-010–induced antimetastatic property. The cause ofG-induced suppression of β-catenin levels is yet toly understood; one possibility is that once β-cateninased into the cytoplasm, it is stabilized by Wnt sig-inacoordinated fashion.TheblockadeofP-cadherin/nin signaling disrupts this process and results in thequent degradation of β-catenin by nonspecifices (43).tenin is well known for controlling a wide array ofr functions. High levels of nuclear β-catenin havefrequently observed in cells with β-catenin or APCmutations (44). We were unable to detect nuclearnin levels in the cell lines used in this report, includ-DA-MB-435-CDH3, PC3-CDH3, and DU145, possi-e to their functional (wild-type) β-catenin and APCns. One noteworthy observation is that, althoughllular distribution of β-catenin was primarily de-on the membrane, nuclear β-catenin remains tran-ionally active, which is consistent with previousgs (45). The supporting data are that the decreasembrane β-catenin by PF-03732010 not only resultedinished levels of vimentin (Fig. 5A) but also the re-n in other β-catenin transcription-mediated targetins (Fig. 5D), including cyclin D1, survivin, and(46, 47). Cyclin D1 mediates tumor cell proliferationrvival, and Bcl-2 prevents tumor cells from undergo-optosis. These observations provide evidence that,ition to the reduced metastatic potential, suppres--catenin levels by PF-03732010 also resulted in anti-erative activity and apoptosis in tumor cells. Therrent decrease in β-catenin and Ki67 levels, as wellincrease of caspase-3 staining, further confirmed

ological relevance of a β-catenin signaling blockadeRece

hesion system in signaling and cancer. J Clin Invest 2002;109:–91.ty RA, Steinberg MS. Cadherin-mediated cell-cell adhesion and

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antiproliferative activity of PF-03732010 was fur-ssessed by using the hybrid [18F]FLT-PET/CT imag-n the MDA-MB-435HAL-CDH3 SRC model. Inment with the results from Ki67 immunohisto-ical staining, PF-03732010 significantly (P < 0.05s vehicle group) suppressed [18F]FLT uptake inprimary and secondary tumors, indicative of anti-erative activity as a downstream effect of blockingherin/β-catenin signaling. The results also suggest18F]FLT-PET imaging may be used in early clinicalpment of PF-03732010 as a supplementary surro-iomarker. Comparing to the conventional biopsyach, the imaging technology in some cases pre-advantages due to the noninvasive nature and lessgeneity. In the clinical setting, showing the suffi-reduction in [18F]FLT uptake in patients on treat-of PF-03732010 at biologically effective dosesconstitute proof of mechanism (48).

en together, these data highlight the critical role ofherin/β-catenin signaling in regulating tumor celltasis, proliferation, and apoptosis. PF-03732010nizes P-cadherin signaling and leads to the antitu-nd antimetastatic efficacy in target-associated tumorls without introducing noticeable adverse effect. Thisprovides the rationale and guidance for the clinicalpment of PF-03732010, in which tumors with highherin expression will be essential criteria for patienton. Future work is warranted to seek a refined ther-ic strategy for patient stratification and combinationies, aiming to reach the full potential for clinical de-ment of the IgG. PF-03732010 is currently under1 trial development.

osure of Potential Conflicts of Interest

authors are current or former employees and shareholders ofInc.

owledgments

thank many Pfizer colleagues for their contributions to this work,ng P-cadherin project team, Bioimaging CoE team, Investigationalgy in Drug Safety Research and Development, Pfizer Worldwiderative Medicine La Jolla Division, and Protein Therapeutics CoE

costs of publication of this article were defrayed in part by thet of page charges. This article must therefore be hereby markedsement in accordance with 18 U.S.C. Section 1734 solely tothis fact.

ived 05/18/2010; revised 08/11/2010; accepted 08/16/2010;
he PF-03732010–induced antitumor activity. published OnlineFirst 09/09/2010.
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2010;16:5177-5188. Published OnlineFirst September 9, 2010.Clin Cancer Res Cathy C. Zhang, Zhengming Yan, Qin Zhang, et al. P-Cadherin with Antitumor and Antimetastatic ActivityPF-03732010: A Fully Human Monoclonal Antibody against

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