full text (pdf) - clinical cancer...

Cancer Therapy: Preclinical

Preclinical Development of an Anti-NaPi2b(SLC34A2) Antibody–Drug Conjugate as aTherapeutic for Non–Small Cell Lung and OvarianCancersKedan Lin, Bonnee Rubinfeld, Crystal Zhang, Ron Firestein, Eric Harstad, Leslie Roth,Siao Ping Tsai, Melissa Schutten, Keyang Xu, Maria Hristopoulos, and Paul Polakis

Abstract

Purpose:Antibody–drug conjugates (ADC) selectively deliver acytotoxic drug to cells expressing an accessible antigenic target.Here, we have appendedmonomethyl auristatin E (MMAE) to anantibody recognizing the SLC34A2 gene product NaPi2b, thetype II sodium–phosphate cotransporter, which is highly expres-sed on tumor surfaces of the lung, ovary, and thyroid as well asonnormal lungpneumocytes. This study evaluated its efficacy andsafety in preclinical studies.

Experimental Design: The efficacy of anti-NaPi2b ADC wasevaluated in mouse ovarian and non–small cell lung cancer(NSCLC) tumor xenograft models, and its toxicity was assessedin rats and cynomolgus monkeys.

Results: We show here that an anti-NaPi2b ADC iseffective in mouse ovarian and NSCLC tumor xenograft

models and well-tolerated in rats and cynomolgus monkeysat levels in excess of therapeutic doses. Despite high levels ofexpression in normal lung of non-human primate, the cross-reactive ADC exhibited an acceptable safety profile with adose-limiting toxicity unrelated to normal tissue targetexpression. The nonproliferative nature of normal pneumo-cytes, together with the antiproliferative mechanism ofMMAE, likely mitigates the potential liability of this normaltissue expression.

Conclusions: Overall, our preclinical results suggest thatthe ADC targeting NaPi2b provides an effective new therapyfor the treatment of NSCLC and ovarian cancer and is current-ly undergoing clinical developments. Clin Cancer Res; 21(22);5139–50. �2015 AACR.

IntroductionIn the United Sates, non–small cell lung cancer (NSCLC) is the

most common cause of cancer death andovarian cancer is thefifthmost common cause of cancer mortality in women (1). Despitethe success of targeted therapies, such as erlotinib (Tarceva) (2)and bevacizumab (Avastin) (3) in lung cancer, and sensitivity toplatinum-based therapy in ovarian cancer, recurrence or progres-sion following initial treatment occurs in the majority of patientsin both diseases. In addition, standard-of-care therapies are asso-ciated with significant systemic toxicity. Treatments that providemeaningful clinical benefit with acceptable safety profiles remaina significant unmet need for NSCLC and ovarian cancers.

Antibody–drug conjugates (ADC) offer the potential to deliverhighly potent cytotoxic agents to cells expressing a predefined cell-surface target. The recent success with ADCs targeting erbB2 inbreast cancer (trastuzumab emtansine, T—DM1) and CD30 inlymphoma (brentuximab vedotin) have validated this overall

approach in oncology applications (4–6). The availability ofhigh-throughput gene expression data has enabled the identifi-cation of new potential ADC targets that are highly expressed oncancer cells but exhibits restricted distribution in normal tissues.In our search for potential ADC targets for the treatment ofNSCLC, we identified SLC34A2, which codes for the type IINa/Pi cotransporter, NaPi2b. NaPi2b is a multitransmembrane,sodium-dependent phosphate transporter (7), which is expressedin human lung, ovarian, and thyroid cancers (8, 9). As a memberof the SLC34 solute carrier protein family (10), it is responsible fortranscellular inorganic phosphate absorption andmaintenance ofphosphate homeostasis (7, 11, 12) and has been associated withcell differentiation (13) and tumorigenesis (14). The differentialexpression in tumor relative to most normal tissues, cell-surfacelocalization, and endocytosis makes it a promising target for ADCtherapeutics.

In contrast to an ideal ADC target with negligible normal tissueexpression, NaPi2b is expressed at a detectable level in normallung tissues, where it takes part in the maintenance of local Piconcentration (7, 10, 15–19). Expression of NaPi2b in normaltissues could represent a potential safety concern for ADC therapy.For example, expression of the target glycoprotein nonmetastaticmelanoma protein B (GPNMB) on normal human skin resultedin adverse dermatologic events in clinical trials of an anti-GPNMBantibody armed with the anti-mitotic monomethyl auristatin E(MMAE) (20). Targeting normal tissues, particularly those withbasal regenerative and proliferative activities, with microtubule-disrupting chemotherapies, should be avoided. Normal lung

Genentech Research and Early Development, South San Francisco,California.

K. Lin and B. Rubinfeld contributed equally to this article.

Corresponding Author: Paul Polakis, Genentech, Inc., 1 DNA Way, South SanFrancisco, CA 94080. Phone: 650-225-5327; Fax: 650-225-5300; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-14-3383

�2015 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org 5139

on April 19, 2018. © 2015 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 8, 2015; DOI: 10.1158/1078-0432.CCR-14-3383

http://clincancerres.aacrjournals.org/

cells, however, in the absence of injury, are not highly proliferativeand therefore less susceptible to antimitotic agents such asMMAE,which largely relies upon cell division to elicit cytotoxic effects.This consideration, together with the retention of high levels ofNaPi2b on lung cancer cells, as well as its overexpression onovarian cancers, compelled us to assess it as an ADC target.

Anti-NaPi2b–vc–MMAE is an ADC composed of a humanizedIgG1 anti-NaPi2b monoclonal antibody (mAb) and MMAE,linked through a protease sensitive valine–citrulline peptide. Thelinker is designed for cleavage by cathepsins to release drugsfollowing endocytotic uptake into lysosomes (21–23). The drug,MMAE, which is a synthetic analog of the antimitotic agentdolostatin 10, binds to and blocks the polymerization of tubulin.Here, we describe the generation and preclinical characterizationof anti-NaPi2b–vc–MMAE and demonstrate its potential as atreatment for NSCLC and ovarian cancers.

Materials and MethodsmRNA tissue expression analysis

The analysis of NaPi2b mRNA expression in multiple humantumor and normal biopsy samples was conducted with micro-array data from Gene Logic Inc. The analysis shown for probe setID 204124 was conducted using the GeneChip Human GenomeU133 Plus 2.0 array on 3,879 normal human tissue samples,1,605 human cancer tissue samples (1,291 primary and 314metastatic), and 3,872 human noncancer disease tissue samples.Microarray data were normalized using the Affymetrix MicroarraySuite version 5.0 software with sample expression values scaled toa trimmed mean of 500.

NaPi2b expression in malignant and normal tissuesdetermined by immunohistochemistry

The reactivity of the mouse anti-NaPi2b antibody on humanlung carcinomas (31 adenocarcinomas and 25 squamous cellcarcinomas), ovarian carcinomas (67 ovarian tumors), and thy-roid carcinomas (56 thyroid carcinomas, adenomas, and thyroidhyperplasias) was assessed by immunohistochemical (IHC) anal-ysis. NaPi2b expression in normal human and monkey tissueswas also evaluated using a normal human tissue microarray ornormal monkey tissues stained with the mouse anti-NaPi2bantibody. IHC was performed on 5-mm-thick formalin-fixed,

paraffin-embedded tissue sectionsmounted on glass slides. Slideswere deparaffinized in xylene and rehydrated through gradedalcohols to distilled water. Slides were pretreated with TrilogyPretreatment Solution (Cell Marque) for 1 hour at 99�C and thentreated with blocking solution (Kierkegaard and Perry Laborato-ries) and avidin/biotin block (Vector Laboratories) respectively.Nonspecific IgG binding was blocked using TBS with Tween-20containing 1% BSA (Roche) and 10% normal horse serum (LifeTechnologies Corp.). Primary antibody, mouse anti-humanNaPi2b, was incubated on slides at 10 mg/mL for 60 minutes atroom temperature. Slides were rinsed and then incubated withhorse anti-mouse biotinylated secondary followed by incubationin Vectastain ABC Elite reagent (Vector Laboratories). Slides werethen incubated in Pierce metal enhanced diaminobenzidine(Thermo Fisher Scientific), counterstained, dehydrated, andcover-slipped.

NaPi2b cloningA human NaPi2b sequence (GNE DNA 393084, encoded by

NM_006424) was subcloned into pRK5tkneo (GNE) cytomega-lovirus mammalian expression, and a stable cell line was gener-ated in human embryonic kidney (HEK) 293 under 200 mg/mLneomycin selection (293 NaPi2b B8).

mAb and ADC productionMousemAbs toNaPi2bwere generated by immunizing BALB/c

mice with recombinant human NaPi2b poly-His–tagged protein(amino acids 250–361). Immunization, hybridoma selection,antibody humanization, and purification processes were doneby the same methodology as described previously for MUC16(24). The 2 control antibodies, anti-6-transmembrane epithelialantigen of prostate 1 (anti-STEAP1) and anti-glycoprotein (anti-gD) mAbs, were constructed as previously described (25, 26). Allantibodies were conjugated with MMAE via a protease labilelinker, MC-VC-PAB as described previously (22). The averagedrug-to-antibody ratio is 3.5 for anti-Napi2b conjugate and othercontrol conjugates.

Cell linesHEK 293 cells (CRL-1573) were transfected with a pRK5tkneo

cytomegalovirusmammalian expression vector (Genentech) con-taining human NaPi2b, and a stable cell line (B8) was generatedin 50:50 DMEM and Ham F-12 media (Genentech) supplemen-ted with 10% FBS, 2 mmol/L L-glutamine, 1� penicillin–strep-tomycin, and 200 mg/mLG418, at 37�C in 5%CO2. TheOVCAR3ovarian cancer mammary fat pad (MFP) transplant xenografttumor model, C93-071405, was developed using the OVCAR3ovarian adenocarcinoma cell line, which was obtained fromATCC. The OVCAR3-X2.1 cell line was derived from an OVCAR3human ovarian adenocarcinoma MFP transplant line (OVCAR-3MFPNo. 4382-061404) developed from theOVCAR3 (HTB-161)cell line from theATCC. TheNCI-H441 (HTB-174), a human lungadenocarcinoma cell line, was obtained from ATCC; Igrov-1, ahuman ovarian adenocarcinoma cell line, was obtained from theNCI cell collection.

Binding affinity by biacore and in vitro cellular staining byimmunoblotting

Binding activity of the anti-NaPi2b mAb was determined inmultiple NaPi2b-expressing cell lines. Anti-NaPi2b immunoblot

Translational Relevance

Non–small cell lung cancer (NSCLC) is the most commoncause of cancer death, and ovarian cancer is the fifth mostcommon cause of cancer mortality in women in the UnitedStates. Here, we describe an anti-NaPi2b antibody–drug con-jugate (ADC), which specifies the delivery of a cytotoxicmonomethyl auristatin E (MMAE) to cells expressing theSLC34A2 gene product NaPi2b, the type II sodium–phosphatecotransporter. We demonstrate that an anti-NaPi2b ADC iseffective in mouse ovarian and NSCLC tumor xenograft mod-els and well-tolerated in rats and cynomolgus monkeys atlevels in excess of therapeutic doses. Overall, our preclinicalresults suggest that the ADC targeting NaPi2b provides aneffective new therapy for the treatment of NSCLC and ovariancancer and is currently undergoing clinical developments.

Lin et al.

Clin Cancer Res; 21(22) November 15, 2015 Clinical Cancer Research5140




of cell lysates from PC3 cells, expressing the SLC34A cDNA orvector control, and 2 ovarian cancer cell lines (OvCAR3 andIgrov1) expressing the endogenous SLC34A gene were pretreatedwith or without deglycosylase (�/þ) to investigate the impact ofglycosylation on binding to antibody.

Determination of in vitro binding by flow-cytometry assayBinding of anti-NaPi2b mAb and ADC to OVCAR3-X2.1, 293

NaPi2b B8, and a nonexpressing cell line was analyzed by flowcytometry as previously described (24). The FACS buffer wasprepared with PBS plus 1% FBS and 2 mmol/L EDTA.

TheOVCAR3-X2.1 tumor tissues were harvested, chopped, andmacerated with cell dissociation buffer (Gibco-BRL; catalog No.13151-014; Invitrogen). Dissociated tumors were incubated at37�C for 15 minutes and then poured and washed through thestrainer with FACS buffer. All subsequent steps were carried out at4�C. Cells were spun down and resuspended with 20 mL ofammonium chloride-potassium red blood cell lysis buffer (Gen-entech; 163 mmol/L NH4Cl, 10 mmol/L KHCO3, 0.1 mmol/LEDTA, pH 7.4) to lyse the red blood cells. Samples were spundown at 4�C and resuspended in FACS buffer and incubated for 1hour each with 3 mg/mL of primary antibodies and then stainedwith the phycoerythrin-labeled anti-human IgG Fc-specific sec-ondary antibody at 2 mg/mL.

All samples were stained with propidium iodide at 0.5 mg/mLand then analyzedusing a FACSflowcytometer (FACSCalibur; BDBiosciences). Cytometric data were processed using the FlowJosoftware application (version 8.4.5) and selection for viable cellswas made on the basis of propidium iodide exclusion forward-scatter values. Geometric mean fluorescence intensity values foreach gated populations were transferred to Excel (version 11.5.6;Microsoft) for graphic display.

Internalization of anti-NaPi2b mAb and ADC by fluorescencemicroscopy

NaPi2b-negative 293 cells, NaPi2b-positive 293 NaPi2b B8cells, andOVCAR3-X2.1 cells were seeded onto BD BioCoat poly-D-lysine–treated cell culture chamber slides, and the slides werecultured at 37�C in a humidified incubator chargedwith 5%CO2.Twenty-four hours later, cell culture media were replaced withfresh growthmedium containing 3 mg/mL of anti-NaPi2bmAb orADC plus 50 mg/mL leupeptin and 5 mg/mL pepstatin to inhibitlysosomal degradation. After 20-hour incubation, cells werewashed and then fixed with 4% formaldehyde in PBS. Afterfixation, cells were washed with PBS and permeabilized with0.05% (w/v) saponin in PBS and then incubated with 1% BSAin PBS to block nonspecific bindings. Lysosomes were thenlabeled with 2 mg/mL mouse anti-lysosomal–associated mem-brane protein 1 (LAMP1)mAb (BD Biosciences), followed by theaddition of 2 mg/mL Alexa488 goat anti-mouse IgG antibody foranti-LAMP1 antibody detection and 2 mg/mL Cy3 goat anti-human IgG antibody for anti-NaPi2b ADC detection. After wash-ing, chamber inserts were removed to expose the glass slides andslides were mounted by applying VectaShield with 40,6-diami-dino-2-phenylindole to label nuclei (Vector Laboratories). Theslideswereplacedunder a glass coverslip and sealedwith clear nailpolish. Images were acquired on a Nikon TE-300 inverted micro-scope equipped with a 60� magnification 1.4 NA infinity-cor-rected PlanApo oil objective (Technical Instruments) and a RetigaEX-cooled CCD camera (Q Imaging), using the QCapture (ver-sion 3.1.1) software application (Q Imaging). Image analysis was

performed using Photoshop CS software (version 8.0; AdobeSystems).

Inhibition of in vitro cell proliferationThe effects of anti-NaPi2b mAb and ADC and free MMAE on

tumor cell viability were assessed using Cell Titer-Glo assay(Promega Corp.) as previously described (27). HumanNaPi2b-positive 293-B8 and NaPi2b-negative 293 cells wereseeded at 5,000 cells, and OVCAR3-X2.1 cells were seeded at2,000 cells per well with 50 mL culture medium in 96-well cultureplates. Anti-NaPi2b ADC was serially diluted and added into thewells. After 4 days, Cell Titer-Glo reagent was added into the plate.The luminescent signal was measured using a Wallac Victor2-V1420 Multilabel HTS Counter (PerkinElmer) and the data wereanalyzed using Excel (version 11.3.7; Microsoft Corp.). Fiftypercent inhibitory concentration (IC50) values were determinedon the basis of cell viability values calculated as percentage ofuntreated control using KaleidaGraph 3.6 (Synergy Software)4-parameter sigmoidal fit. The same procedure was used in theanalysis of a nontarget-specific control, anti-gDADCat 2.4mg/mLto address the target specificity in antiproliferation activities.

In vivo efficacyAll efficacy studies were conducted in accordance with the

Guide of the Care and Use of laboratory Animals (28).For the OVCAR3-X2.1 efficacy study, the cell line was derived

from an OVCAR3 ovarian adenocarcinoma transplant lineobtained from ATCC (24). OVCAR3 cells were injected intraper-itoneally into female C.B-17 severe combined immunodeficient(SCID).beige mice. A donor tumor was excised from a mousebearing intraperitoneal tumors,minced, and surgically implantedinto the right thoracic mammary fat pad (MFP) of female C.B-17SCID.beige recipientmice. The tumors were serially passaged intothe right thoracicMFPof recipientmice tomaintain the transplantline. Female SCID or beige with x-linked immunodeficient micefromCharles River Laboratories were inoculated in the 2 of 3MFPwith 50�106OVCAR3-X2.1 or 25�106 Igrov1 cells, respectively,suspended inHankbalanced salt solutionwithMatrigel. TheNCI-H441 efficacymodel was donewith an injection of 50� 106 NCI-H441 cells into the right dorsal flank in C.B-17 SCID.beige mice,suspended inHank balanced salt solutionwithMatrigel. Themicewere 6- to 12 weeks old and weighed approximately 20 to 25 geach. When tumors reached a volume range of 100 to 300 mm3,animals were randomized into groups of 6 to 10 each and dosedwith vehicle control or anti-NaPi2b ADC at a dose range of 3 to 24mg/kg. MMAE conjugated to anti-gD antibody was also includedas a control.

Tumors were measured using UltraCal-IV calipers (Model 54-10-111, Fred V. Fowler Company) according to the followingformula: tumor volume (mm3) ¼ (length � width2) � 0.5. Micewere euthanized before tumor volume reached 3,000 mm3 orwhen tumors showed signs of impending ulceration.

In vivo pharmacokinetic studiesAll studies were conducted in accordance with the Guide of the

Care and Use of Laboratory Animals (28). To fully characterizethe pharmacokinetic profiles of anti-NaPi2b ADC, single dosestudies were conducted in OVCAR3-X2.1 tumor–bearing andnontumor female SCID.beige mice from Charles River Laborato-ries, Sprague-Dawley (SD) rats from Charles River Laboratories,

Preclinical Development of NaPi2b Antibody–Drug Conjugate

www.aacrjournals.org Clin Cancer Res; 21(22) November 15, 2015 5141




and cynomolgus monkeys (Macaca fascicularis) from SNBL USALtd. The mice were 6 to 8 weeks old and weighed approximately16 to 28 g; the rats were 6 to 13 weeks old and weighedapproximately 160 to 450 g; and the monkeys were 3 to 8 yearsold and weighed approximately 2 to 7 kg. Anti-NaPi2b ADC wasadministered intravenously in tumor-bearing mice (1, 3, and 6mg/kg), non—tumor-bearing mice (0.5 and 5 mg/kg), rats (0.5and 5 mg/kg), and monkeys (0.3 and 1 mg/kg). Blood sampleswere collected and analyzed for total antibody concentrations byELISA. Pharmacokinetic parameters were estimated using a 2compartmental model with WinNonlin (version 5.2.1, PharsightCorporation).

ELISA for total antibody concentration measurementMultiple ELISA formats were used to analyze total antibody

concentrations in different matrices. A bridging format ELISA was

developed for samples frommice (both tumor-bearing and non–tumor-bearing) and rats; the assay used a biotinylated recombi-nant humanNaPi2b extracellular domain protein and a goat anti-human IgG conjugated to horseradish peroxidase (HRP; BethylLaboratories, Inc.) to capture anti-NaPi2b antibodies. Thesereagents were coincubated with diluted samples, standards, orassay controls and then added to a NeutrAvidin-coated plate forcapturing the reagent-analyte complex. The lower limit of quan-tification (LLOQ) for the assay is <0.313 ng/mL with a minimum1:100 dilution. The monkey serum samples were analyzed with asimilar bridging format by using a sheep anti—human IgGconjugated to biotin and HRP (The Binding Site Inc.) to captureanti-NaPi3b antibodies. The reagent–analyte complex is capturedin streptavidin-coated plates followed by the color developmentreaction. The assay has a LLOQof 0.100 mg/mL (0.660 nmol/L) incynomolgus monkey serum.

Figure 1.Expression of NaPi2b by transcript in human tissues. A, analysis of mRNA transcript. Measurements were carried out on the Affymetrix U133P chip and areexpressed as scaled average difference. Each dot represents a normal (green), tumor (red), or diseased nontumor (blue) human tissue specimen. Rectanglesencompass the 25th to 75th percentile range for each distribution. WBC, white blood cells. B, IHC detection of NaPi2b protein in the indicated tumor samplesrepresenting increasing intensity of staining.

Lin et al.





Toxicity studies in rat and monkeyThe safety profile of anti-NaPi2b ADC was evaluated in na€�ve

SD rats (Charles River Laboratories) and cynomolgus monkeys(M. fascicularis, Covance Research Products Inc.).Male and femalerats (250–450 g) were given intravenous injections of vehicle oranti-NaPi2b ADC at 2, 6, or 12 mg/kg once weekly for 4 weeksfollowed by a 6-week recovery period. Thirty rats (15male and 15female) per group were designated for toxicity evaluations at theend of dosing (day 26, 10/sex/group) and recovery period (day64, 5/sex/group) necropsies. Toxicological assessments includedevaluation of mortality, clinical signs, body weights, food con-sumption, ophthalmic examinations, functional observation bat-tery, motor activity, micronuclei in bone marrow, clinical pathol-ogy, and anatomic pathology.

Male and femalemonkeys (2.0–6.5 kg) were given intravenousinjections of vehicle or anti-NaPi2b ADC at 1, 3, or 6 mg/kg onceevery 3 weeks for 5 doses followed by a 6-week recovery period.Five monkeys per sex per group were designated for toxicityevaluations at the end of dosing (day 92, 3/sex/group) andrecovery period (day 127, 2/sex/group) necropsies, respectively.Two additional monkeys were included in each group for cardio-vascular evaluations via telemetry. Toxicologic assessmentsincluded evaluation of mortality, clinical signs, body weights,food consumption, neurologic and physical examinations, res-piration rates, ophthalmic and electrocardiograms (external ortelemetry, or both) examinations, blood pressure measurements(external), telemetry hemodynamics, intraabdominal body tem-perature, clinical pathology, and anatomic pathology.

Blood samples were also collected for toxicokinetics (TK)analysis of anti-Napi2b ADC total antibody (conjugated andunconjugated), free MMAE, and anti-therapeutic antibody (ATA)levels.

ResultsNaPi2b is highly expressed in lung and ovarian cancers

To identify potential targets for ADC therapy in lung cancer, wesearched a large mRNA transcript database generated by oligo-nucleotide microarray analysis of more than 5,000 human cancerand normal tissue samples. We failed to identify any transcriptscoding for cell-surface proteins highly overexpressed in lungcancer relative to normal lung while maintaining low expressionin other normal vital tissues. On the basis of the assumption that

an antimitotic drug would spare nonproliferating cells, weexpanded the search to include expression in normal lung tissues.Accordingly, we identifiedNaPi2bmRNA, whichwas expressed athigh levels in normal and cancerous lung tissues, as well as inovarian and thyroid tumor samples relative to the vastmajority ofall other normal and cancer tissues analyzed (Fig. 1A). IHCanalysis, using mouse anti-NaPi2b antibodies, demonstrated ahigh frequency of NaPi2b expression in human nonsquamous,NSCLC, nonmucinous ovarian cancer, and papillary thyroidcarcinomas (Fig. 1B and Table 1). Similar staining frequency andintensity were found in matched primary and metastatic carcino-ma samples of lung and ovarian cancers. IHC staining in normalhuman tissues revealed staining in the epithelial compartments ofseveral tissues including lung, bronchus, and kidney (data notshown).

Anti-NaPi2b antibody and its ADC show specific andcomparable binding affinities to human and nonhumanprimate

NaPi2b is a cell-surface transporter containing 12 transmem-brane domains with both the amino and carboxyl termini local-ized to the cytosol (10). As an initial step toward generating anADC reactive with NaPi2b on live cells, we immunized mice witha recombinant fusion protein containing amino acid sequencepresent in the large extracellular loop separating transmembranedomains 4 and 5. We selected one mAb, hereafter designated asanti-NaPi2b, based on its affinity and cross-reactivity withNaPi2bhomolog expressed by the nonhuman primate cynomolgusmonkey. By immunoblotting, anti-NaPi2b recognized a 100-kDaprotein expressed by PC3 cells transfected with the NaPi2b cDNA(PC3-SLC34A) but not with vector control (Fig. 2A). Ovariancancer cells OVCAR3 and Igrov1 also expressed a similarly sizedendogenous protein reactive with the antibody. Following enzy-matic deglycosylation, the mobility of the NaPi2b proteinincreased but remained reactive with anti-NaPi2b. Anti-NaPi2balso reacted with all 3 positive cell lines as determined by flowcytometry and immunofluorescent detection on live cells (Fig.2B) but notwith the vector control PC3 cell line (data not shown).Thus, anti-NaPi2b recognizes epitopes within the extracellulardomain of NaPi2b independent of glycosylation.

Using a series protein fragments derived from the 111-aminoacid extracellular loop, the reactive epitope was delineated to

Table 1. NaPi2b IHC in lung NSCLC, ovarian carcinoma, and thyroid carcinoma (prevalence %)

Negative 1þ 2þ 3þ Number of cases

Lung NSCLCAdenocarcinoma 13% 10% 35% 42% 31Squamous cell carcinoma 80% 8% 12% 0% 25

Ovarian carcinomaSerous adenocarcinoma 4% 4% 69% 23% 26Mucinous carcinoma 60% 0% 30% 10% 10Endometrioid carcinoma 5% 0% 70% 25% 20Clear cell carcinoma 0% 18% 64% 18% 11

Thyroid carcinomaPapillary carcinoma 9% 9% 48% 34% 23Medullary carcinoma 91% 9% 0% 0% 11Poorly differentiated 64% 0% 27% 9% 11Follicular adenocarcinoma 100% 0% 0% 0% 3Adenoma 100% 0% 0% 0% 2Hyperplasia 100% 0% 0% 0% 6

NOTE: Staining intensity takes into account both intensity of staining and the observation of staining in >50% of the tumor cells where negative, no detectable signal;1þ, weak signal; 2þ, moderate signal; and 3þ, strong signal.






sequence contained between amino acid positions 320 to 361 offull-length human NaPi2b sequence (Fig. 3A). Appropriate safetyassessment of ADCs requires testing in a species with targetantigen that is cross-reactive with the therapeutic antibody. Acomparison of the amino acid sequence corresponding to posi-tions 320 to 361 of human NaPi2b revealed 7 differences relativeto the rat homolog, but only one difference in the cynomolgusmonkey sequence (Fig. 3B). Anti-NaPi2b did not react with cellsexpressing the rat NaPi2b cDNA (data not shown). However, at

concentrations of antibody ranging from 0.1 to 10 mg/mL, cellsexpressing the cynomolgus monkey cDNA were positive by flowcytometry with signal intensity equivalent to that of cells expres-sing human cDNA (Fig. 3B). Immunoblotting of a series ofhuman NaPi2b fragments containing single amino acid substitu-tions revealed a loss of reactivity upon substitution of a leucinewith a tyrosine at position 327 (L327Y, Fig. 3C). This replacementreflects the corresponding residue contained in rat NaPi2b and isconsistent with the lack of recognition of rat NaPi2b by ourantibody. In contrast, reactivity was largely retained with theN335T mutant, which reflects the single alteration in the cyno-mologus monkey sequence. Binding affinity of anti-NaPi2bantibody to human and cynomolgus monkey NaPi2b was com-parable by radioligand cell-binding assays, with Kd values ofapproximately 10.19� 0.74 and 8.42� 0.81 nmol/L, respective-ly. Comparable expression of NaPi2b in human and cynomolo-gus monkey pulmonary bronchus and alveoli was demonstratedby IHC staining of lung tissues with anti-NaPi2b (Fig. 3D).

Anti-NaPi2b ADC is internalized effectively and displays strongin vitro activity

The potent cytotoxic compoundMMAEwas conjugated to anti-NaPi2b by using a linker containing the protease-sensitive vcdipeptide (22). The resulting anti-NaPi2b ADCbound to live cellsas effectively as the unconjugated antibody as determined by flowcytometry (data not shown). Following cell-surface binding, theactivation of the ADC requires efficient internalization and traf-ficking to degradative lysosomal vesicles. We monitored theuptake and internalization of anti-NaPi2b ADC following a 20-hour incubation with live cells that were then fixed and permea-bilized for immunofluorescent detection. Localization of lyso-somes was determined using a mouse IgG antibody againstLAMP1 and a fluorescent secondary antibody against mouse IgG.Anti-NaPi2bADCwas readily detected in LAMP1-positive vesiclesin 293 cells overexpressing NaPi2b but not in the 293 vectorcontrol cell line (Fig. 4A). Uptake and trafficking to LAMP-1–positive vesicles was also observed in ovarian cancer cell linesOVCAR3 and Igrov1, which expressedNaPi2b endogenously (Fig.4B). We also tested the NCSLC cell line NCI-H441; however, thelow level of endogenous NaPi2b expressed by this cell lineprecluded its detection in LAMP-1–positive vesicles. These resultsdemonstrate the delivery of anti-NaPi2b ADC to lysosomeswhereMMAE or MMAE-containing catabolites may be released to exertcytotoxicity, consistentwith the proposedmechanismof action ofADCs (29).

To evaluate target-dependent cell killing with anti-NaPi2bADC, the293 cell line that overexpressedNaPi2band thematchedvector control cell line were treated with increasing amounts ofADC. No loss of viability was observed for the control cell line atconcentrations of ADC exceeding 10 mg/mL, whereas the 293-NaPi2b cell line exhibited a dose-dependent reduction in viabil-ity, with an IC50 of approximately 0.15 mg/mL (Fig. 5A). We nexttested the cancer cell lines that expressed NaPi2b endogenously.Strong cell-surface expression of NaPi2b was observed with theovarian cancer cell lines OVCAR3-X2.1 and Igrov1, whereasexpression in the NCI-H441 lung cancer cell line was significantlylower (Fig. 5B). Although comparable amounts of NaPi2b wereexpressed on the cell surface of the OVCAR3 and Igrov1 cell lines,the OVCAR3 appeared to be more sensitive to the ADC (Fig. 5C).Commensurate with its low level of NaPi2b expression, the NCI-H441 lung cancer cell linewas far less sensitive to the ADC relative

Figure 2.Binding activity of the anti-NaPi2b mAb. A, anti-NaPi2b immunoblot of celllysates from PC3 cells expressing the SLC34A cDNA or vector control and 2ovarian cancer cell lines (OvCAR3 and Igrov1) expressing the endogenousSLC34A gene. Sampleswere pretreatedwith or without deglycosylase (�) asindicated. B, flow cytometry of OVCAR3-X2.1 cells. Live cells were subjectedto flow-cytometric (left) or immunoflourescent (right) analysis with theNaPi2b antibody. The red, green, and blue lines indicate incubation withoutantibody, secondary antibody only, or both primary and secondaryantibodies, respectively.

Lin et al.





to the ovarian cells. For all 3 of the cell lines, the control ADCwasineffective at concentrations below 5 mg/mL.

Anti-NaPi2b ADC inhibits tumor growth and causes tumorregression in animal models

Three xenograft tumor models, OVCAR3-X2.1 and Igrov1 deriv-ed fromhumanovarian cancer andNCI-H441derived fromhumanlung adenocarcinoma epithelial cell, were used in the in vivo efficacystudies. Animals received a single intravenous injection of vehiclecontrol, anti-NaPi2b ADC, or dose-matching ADC control at theindicated dose levels. All 3 models demonstrated specific and dose-dependent inhibitory activity compared with the vehicle control(Fig. 6). Despite the apparent lower level of antibody uptake and

cell-surface expression observed in vitro (Figs. 4B and 5B), sensitivityof the NCI-H441 tumors was similar to that of OVCAR3 X2.1model. This could be explained by the robust and comparablelevels of NaPi2b detected by staining tumor sections obtained fromtheNCI-H441 andOVACAR3�2.1models (Fig. 6 insets).However,the Igrov1 tumor expressed levels of NaPi2b similar to the other 2models but was relatively less sensitive to the ADC, indicating thatfactors in addition to ADC target level contribute to the response.At the same dose level, anti-NaPi2b ADC showed a substantiallygreater tumor growth inhibition than the ADC control, indicatingtargeted antitumor activity of anti-NaPi2b ADC. These resultssuggest that efficacy can be achieved with appropriate dosing ofanti-NaPi2b ADC in a variety of tumor xenograft models.

Figure 3.Characterization of the NaPi2bantibody. A, indicated fragments ofthe NaPi2b were fused to GST andsubjected to SDS-PAGE and stainedwith Coomassie blue (left) orimmunoblotted with anti-NaPi2b. B,cells transfected with either human(blue line) or cynomolgus monkey(green line) Napi2b cDNA wereanalyzed by flow cytometry withincreasing concentrations of anti-NaPi2b or secondary antibody alone(red line). The relevant amino acidsequences of human, rat, andcynomolgus are presented forcomparison. C, indicated single aminoacid substitutions were introducedinto the human NaPi2b sequence andthe resulting proteins analyzed byimmunoblotting with anti-NaPi2b. D,immunohistochemical detection ofNaPi2b in lung tissue from humanand cynomolgus monkey withanti-NaPi2b.






Anti-NaPi2b ADC exhibits linear pharmacokinetics in rodentand nonhuman primate studies

To further understand the relationship between systemic expo-sure and antitumor efficacy, the pharmacokinetic property of anti-NaPi2b ADC in SCID.beige mice bearing OVCAR3-X2.1 tumorswere evaluated. Consistent with the in vivo efficacy studies dis-cussed earlier, dose-dependent efficacy measured by tumor vol-ume was observed for animals administrated with anti-NaPi2bADC at 1, 3, and 6 mg/kg (data not shown). Pharmacokineticanalysis of total antibody, which includes both the anti-NaPi2bADCand the unconjugated antibody, demonstrated linearity overdoses of 1 to 6 mg/kg. In tumor-bearing mice dosed with anti-NaPi2b ADC at 0.5 and 5 mg/kg, the clearance ranged from 9.09to 10.8 mL/kg/d and was comparable with the range of 8.94 to9.13 mL/kg/d in non—tumor-bearing mice dosed identically.This indicates that antigen expressed on tumors did not impactthe pharmacokinetics (Fig. 7 and Table 2).

Figure 4.Cell internalization of NaPi2b antibody. Live cells were incubated with2 mg/mL of anti-NaPi2b antibody for 2 hours at 37�C and then fixed andcostained with antibody to LAMP1 to localize lysosomes. Anti-NaPi2b wasdetected with Cy3-labeled secondary antibody (red) and antibody to LAMP1with Alex488-labeled secondary antibody (green). Only the merged imagesare presented in color (color overlay). A, 293 cells stably expressing theSLC34A cDNA (bottom) or vector control (top). B, cancer cell linesendogenously expressing the SLC34A gene.

293-vector293-NaPi2b

500

400

300

200

100

0

OVCAR3 x21

MF

IC

ell v

iabi

lity

(% o

f unt

reat

ed c

ontr

ol)

Igrov1 H441

20

40

60

80

100

120

0.10.01

18

16

14

8

6

4

2

0

0.001 0.01

OVCAR3 ctrlOVCAR3 NaPiIgrov1 ctrlIgrov1 NaPiH441 ctrlH441 NaPi

Antibody (μg/mL)

Lum

ines

cenc

e (R

LU x

10−5

)

0.1 1 10 100

10

12

1.0Antibody (μg/mL)

B

C

A

10 100

Figure 5.Inhibition of in vitro cell proliferation by anti-NaPi2b ADC. Live cells wereincubated with increasing concentrations of the anti-NaPi2b ADC, andviability was assessed by the Cell Titer-Glo assay. A, 293 cells stablyexpressing the SLC34A cDNA or vector control. B, relative intensity of NaPi2bcell-surface expression measured as mean fluorescent intensity (MFI) oncancer cell lines endogenously expressing SLC34A by flow cytometry. C,viability of cancer cells incubated with increasing concentrations of anti-NaPi2b ADC (solid lines) or a non–target-specific control anti-gD ADC(dashed lines).

Lin et al.





Anti-NaPi2b ADC was also tested in rats and cynomolgusmonkeys to fully characterize the pharmacokinetics in preclinicalspecies. The clearance of total antibody was dose-independentafter a single intravenous administration in rats [CL, 21.2 � 5.21and 15.9 (n ¼ 2) mL/kg/d for 0.5 and 5 mg/kg, respectively) orcynomolgusmonkeys (CL, 10.8� 0.804 and 13.8� 2.76mL/kg/d

for 0.3 and 1 mg/kg, respectively) at the dose range studied. Thissuggests that thepresenceof cross—reactive antigen in themonkeyshad no apparent impact on the clearance (Fig. 7 and Table 2).

Anti-NaPi2b ADC was well-tolerated in rats and monkeysThe major effects of anti-NaPi2b ADC included bone marrow

toxicity in rats and monkeys, and liver and testicular toxicityin rats. The observed bone marrow toxicity led to the death of

2,000OVCAR3 x2.1

1,500

1,000

500

00 10 20

Igrov1

H441

V 33

6

6

6

6

12, 24

12

6

12

3

63

V

IgG-vc-MMAEα-NaPi2b-vcMMAE

1224

V

30 40 50

00

500

1,000

1,500

2,000

Fitte

d tu

mor

vol

ume

(mm

3 )Fi

tted

tum

or v

olum

e (m

m3 )

Fitte

d tu

mor

vol

ume

(mm

3 )

0

500

1,000

1,500

2,000

8 16 24 484032Day

56 64

0 4 8 12 242016 28 32

Figure 6.Inhibition of xenograft tumor growth. Xenograft tumor models wereestablished for the indicated cell lines as described in Materials andMethods. Animals bearing established tumors were administered a singledose of either control ADC (green lines) or anti-NaPi ADC (purple lines) atthe indicated milligram per kilogram dose levels. Each inset presentsthe relative level of NaPi2b protein in a corresponding xenograft tumorsection as detected by IHC staining.

0 423528Time (days)

21147

0 3528Time (days)

21

Monkey 1 mg/kgMonkey 0.3 mg/kg

147

0 423528

Rat 5 mg/kgRat 0.5 mg/kg

Time (days)2114

0.01

0.1

1

10

Con

cent

ratio

n (μ

g/m

L) 100

1,000

0.01

0.1

1

10

Con

cent

ratio

n (μ

g/m

L) 100

1,000

0.01

0.1

1

10

Mouse 5 mg/kgMouse 0.5 mg/kgTB Mouse 6 mg/kgTB Mouse 3 mg/kg

Con

cent

ratio

n (μ

g/m

L)

TB Mouse 1 mg/kg

100

1,000

7

Figure 7.Anti-NaPi2b-MMAE exhibited linear pharmacokinetics in nonclinical species.Animals were given a single intravenous injection of the ADC at the indicateddoses inmouse (including tumor-bearingmouse, tumor-bearingmouse; top),rat (middle), and monkey (bottom). The concentrations of total antibodywere measured, and average concentrations with SDs were determined from2 to 4 animals per group.






1monkey and 3 rats in the high-dose groups due to neutropenia-related septicemia and marked anemia, respectively. Liver andtesticular effects were primarily noted in epithelial cells, consistingof increased apoptosis and increased arrestedmitotic figures. Theseeffects were largely related to the pharmacology of MMAE as amicrotubule-disrupting agent (i.e., increased apoptotic cells andarrested mitotic figures) and, with the exception of the testiculartoxicity in rats, were reversible after a 6-week post-dose period insurviving animals.Given the durationof the spermatogenic cycle inrats, the persistent effect in male reproductive effects is expected.MMAE-related increases in apoptosis and mitotic figures werenoted in additional tissues; however, theywere ofminimal severityand reversible. A microscopic finding of minimal, chronic-activeinflammation with edema was noted in the lung of 1 male at 6mg/kg. The relationship of this lung finding to the test article isuncertain, as it was seen in a single high-dose animal in a singleregion of the lung, but a test article-related effect cannot be ruledout. Importantly, there were no toxicologically significant micro-scopicfindings present in the recovery animals. Consistentwith thepharmacologic activity of MMAE, doses � 6 mg/kg anti-NaPi2bADC produced an increase in micronucleated polychromatic ery-throcytes in rat bone marrow erythrocytes likely through an aneu-genicmechanism. No toxicologically significant changes in cardio-vascular, respiratory, or neurologic assessment were noted withanti-NaPi2b ADC administration in monkeys or rats. Minor,reversible anti-NaPi2b ADC-related reductions in motor activitycounts were noted in males at 12 mg/kg during the dosing phaseassessment and were consistent with clinical signs of debilitation(i.e., hypoactivity, decreased body weights, stained fur) associatedwith anemia rather than a direct pharmacologic effect.

Exposure to anti-NaPi2b ADC and free MMAE generallyincreased with the increase in dose level in rats and monkeyswith no sex-related differences or accumulation. The increases intotal antibody maximum concentration (Cmax) and the areaunder concentration (AUC) were dose proportional. Severalmonkeys at all doses (11 of 34 total) developed ATAs to anti-NaPi2b ADC; however, these monkeys had similar exposure tothe animals without ATAs.

DiscussionAdvances in ADC technology have resulted in encouraging

responses in recent clinical trials (30). The development of morepotent drugs and linkers with enhanced stability has greatlyimproved the prospects for this approach. This has coincidedwith advances in high-throughput technologies, enabling the

identification of highly specific cell-surface antigens expressed ontumor cells. Our gene expression analysis, in which thousands ofhuman tumor and normal tissue samples were represented,revealed overexpression of NaPi2b mRNA in human lung, ovar-ian, and thyroid cancers. Therefore, we sought to target NaPi2bwith therapeutic antibodies.

Our selection of the anti-NaPi2bmAb for drug conjugationwasbased on high-affinity binding, cross-species reactivity, and a highrate of cellular internalization. When conjugated with MMAEthrough the vc peptide linker, anti-NaPi2b ADC exhibited selec-tive andhigh-affinity binding to human and cynomolgusmonkeyNaPi2b and showed potent and selective inhibition of cell pro-liferation in NaPi2b-expressing cells in vitro. In vivo studies con-firmed that anti-NaPi2b ADC inhibited growth of NaPi2b-expres-sing human ovarian and non-small cell lung tumor xenograftsmodels with single doses of anti-NaPi2b ADC ranging from 3 to24 mg/kg.

Despite high levels of expression in normal lung of nonhumanprimate, the cross-reactive ADC exhibited an acceptable safetyprofile with a dose-limiting toxicity unrelated to normal tissueexpression.Our safety study concluded that anti-NaPi2bwaswell-tolerated in rats up to 12 mg/kg and in monkeys up to 3 mg/kg.The slow proliferative nature of normal pneumocytes, togetherwith the antiproliferative mechanism of MMAE, likely mitigatesthe potential liability of high NaPi2b expression in normal lungtissue. Overall, our preclinical results suggest that the ADC target-ing NaPi2b provides a promise of an effective new therapy for thetreatment of NSCLC and ovarian cancer and is currently under-going clinical developments.

The pharmacokinetics of anti-NaPi2b ADC were fully charac-terized and demonstrated linearity in all the preclinical speciestested, suggesting that NaPi2b expression in normal tissues incynomolgus monkeys as a binding species or in tumor-bearingmice did not show any apparent impact on pharmacokinetics.

The MMAE-conjugated anti-NaPi2b ADC has specific antipro-liferative activity in cancer cells in vitro and antitumor activity inxenograft cancer models expressing NaPi2b. Conjugation ofMMAE to an anti-NaPi2b mAb improved the tolerability andwidened the therapeutic window of MMAE in animal studies.These properties provide the rationale for the selective antitumoractivity of anti-NaPi2b ADC against NaPi2b-expressing tumors.IHC staining in normal human and cynomolgus monkey tissueswas generally consistent and revealed congruous staining in theepithelial compartments of several tissues including lung, bron-chus, and kidney tissues (data not shown). Rats, a nonbindingspecies for anti-NaPi2b ADC, were included in the safety

Table 2. Pharmacokinetic parameters estimated for anti-NaPi2b ADC after intravenous administration in nonclinical species

Anti-NaPi2b total antibodySpecies Dose level,a mg/kg CL, mL/d/kg t1/2, days V1, mL/kg

Tumor-bearing mouse 1 (n ¼ 3) 10.8 6.22 50.33 (n ¼ 3) 10.8 7.68 55.06 (n ¼ 3) 9.09 10.5 51.1

Non–tumor-bearing mouse 0.5 (n ¼ 3) 8.94 12.6 54.85 (n ¼ 3) 9.13 11.5 54.4

Rat 0.5 (n ¼ 3) 21.2 � 5.21 8.43 � 2.39 50.6 � 3.405 (n ¼ 2) 15.9 12.3 51.6

Monkey 0.3 (n ¼ 4) 10.8 � 0.804 8.42 � 1.19 32.5 � 2.341 (n ¼ 4) 13.8 � 2.76 8.26 � 2.15 38.1 � 1.77

NOTE: Determined using a two-compartmental model.Abbreviations: CL, clearance; V1, volume of distribution at central compartment; t1/2, terminal half-life.aNumber of animals per timepoint.


Lin et al.




evaluation to evaluate the antigen-independent toxicity of theconjugate. In normal rats andmonkeys, toxicologically significanteffects were consistent with the pharmacology of MMAE with themost sensitive tissues including bone marrow, liver, and testes.

In summary, anti-NaPi2b ADC has demonstrated significantefficacy in nonclinical NaPi2b-expressing xenograft models andexhibited acceptable safety profiles in animal studies, suggestingthat anti-NaPi2b ADC may be a promising therapeutic for thetreatment of ovarian cancer and NSCLC. With its highly prom-ising results in preclinical studies, anti-NaPi2b ADC has enteredinto clinical development in both ovarian and lung cancers anddemonstrated early evidence of clinical benefit in patients (31).

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: K. Lin, B. Rubinfeld, C. Zhang, E. HarstadDevelopment of methodology: K. Lin, K. Xu

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Lin, B. Rubinfeld, C. Zhang, R. Firestein, E. Harstad,L. Roth, K. Xu, M. HristopoulosAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Lin, C. Zhang, R. Firestein, E. Harstad, S.P. Tsai,M. Schutten, K. Xu, P. PolakisWriting, review, and/or revision of the manuscript: K. Lin, B. Rubinfeld,C. Zhang, E. Harstad, S.P. Tsai, M. Schutten, K. Xu, P. PolakisAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K. Lin, B. Rubinfeld, C. Zhang, S.P. Tsai, K. XuStudy supervision: K. Lin, C. Zhang, E. Harstad, K. Xu

AcknowledgmentsThe authors thank Willy Solis and Hajime Hiraragi for their contributions.

ADC technology was licensed from Seattle Genetics.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 31, 2014; revised May 14, 2015; accepted June 15, 2015;published OnlineFirst July 8, 2015.

References1. Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin

2010;60:277–300.2. Shepherd FA, Rodrigues Pereira J, Ciuleanu T, Tan EH, Hirsh V, Thong-

prasert S, et al. Erlotinib in previously treated non-small-cell lung cancer.N Engl J Med 2005;353:123–32.

3. Sandler A, Gray R, Perry MC, Brahmer J, Schiller JH, Dowlati A, et al.Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lungcancer. N Engl J Med 2006;355:2542–50.

4. Amiri-Kordestani L, Blumenthal GM, XuQC, Zhang L, Tang SW,Ha L, et al.FDA approval: ado-trastuzumab emtansine for the treatment of patientswith HER2-positive metastatic breast cancer. Clin Cancer Res 2014;20:4436–41.

5. de Claro RA, McGinn K, Kwitkowski V, Bullock J, Khandelwal A, Habte-mariam B, et al. U.S. Food and Drug Administration approval summary:brentuximab vedotin for the treatment of relapsed Hodgkin lymphoma orrelapsed systemic anaplastic large-cell lymphoma. Clin Cancer Res 2012;18:5845–9.

6. Tai YT, Mayes PA, Acharya C, Zhong MY, Cea M, Cagnetta A, et al. Novelanti-B-cell maturation antigen antibody-drug conjugate (GSK2857916)selectively induces killing of multiple myeloma. Blood 2014;123:3128–38.

7. Xu H, Bai L, Collins JF, Ghishan FK. Molecular cloning, functional char-acterization, tissue distribution, and chromosomal localization of ahuman, small intestinal sodium-phosphate (Naþ-Pi) transporter(SLC34A2). Genomics 1999;62:281–4.

8. Rangel LB, Sherman-Baust CA,Wernyj RP, Schwartz DR, Cho KR,Morin PJ.Characterization of novel human ovarian cancer-specific transcripts(HOSTs) identified by serial analysis of gene expression. Oncogene 2003;22:7225–32.

9. JarzabB,WienchM, Fujarewicz K, Simek K, JarzabM,Oczko-WojciechowskaM, et al. Gene expression profile of papillary thyroid cancer: sources ofvariability and diagnostic implications. Cancer Res 2005;65:1587–97.

10. Virkki LV, Biber J, Murer H, Forster IC. Phosphate transporters: a tale of twosolute carrier families. Am J Physiol Renal Physiol 2007;293:F643–54.

11. Sabbagh Y, O'Brien SP, Song W, Boulanger JH, Stockmann A, Arbeeny C,et al. Intestinal npt2b plays a major role in phosphate absorption andhomeostasis. J Am Soc Nephrol 2009;20:2348–58.

12. Hilfiker H, Hattenhauer O, Traebert M, Forster I, Murer H, Biber J.Characterization of a murine type II sodium-phosphate cotransporterexpressed inmammalian small intestine. ProcNatl Acad Sci U SA 1998;95:14564–9.

13. Mashima H, Zhang YQ, Tajima T, Takeda J, Kojima I. Na-Pi cotransporterexpressed during the differentiation of pancreatic AR42J cells. Diabetes2001;50 Suppl 1:S39.

14. Kopantzev EP,MonastyrskayaGS, Vinogradova TV, ZinovyevaMV, KostinaMB, FilyukovaOB, et al. Differences in gene expression levels between earlyand later stages of human lung development are opposite to those betweennormal lung tissue and non-small lung cell carcinoma. Lung Cancer2008;62:23–34.

15. Feild JA, Zhang L, Brun KA, Brooks DP, Edwards RM. Cloning andfunctional characterization of a sodium-dependent phosphate transporterexpressed in human lung and small intestine. Biochem Biophys ResCommun 1999;258:578–82.

16. TraebertM,HattenhauerO,MurerH,Kaissling B, Biber J. Expression of typeII Na-P(i) cotransporter in alveolar type II cells. Am J Physiol 1999;277:L868–73.

17. Xu Y, Yeung CH, Setiawan I, Avram C, Biber J, Wagenfeld A, et al. Sodium-inorganic phosphate cotransporter NaPi-IIb in the epididymis and itspotential role in male fertility studied in a transgenic mouse model. BiolReprod 2003;69:1135–41.

18. Frei P, Gao B, Hagenbuch B, Mate A, Biber J, Murer H, et al. Identificationand localization of sodium-phosphate cotransporters in hepatocytes andcholangiocytes of rat liver. Am J Physiol Gastrointest Liver Physiol2005;288:G771–8.

19. Huber K, Muscher A, Breves G. Sodium-dependent phosphate trans-port across the apical membrane of alveolar epithelium in caprinemammary gland. Comp Biochem Physiol A Mol Integr Physiol 2007;146:215–22.

20. Keir CH, Vahdat LT. The use of an antibody drug conjugate, glembatu-mumab vedotin (CDX-011), for the treatment of breast cancer. ExpertOpin Biol Ther 2012;12:259–63.

21. Bai RL, Pettit GR, Hamel E. Binding of dolastatin 10 to tubulin at a distinctsite for peptide antimitotic agents near the exchangeable nucleotide andvinca alkaloid sites. J Biol Chem 1990;265:17141–9.

22. Doronina SO, Toki BE, Torgov MY, Mendelsohn BA, Cerveny CG, ChaceDF, et al. Development of potent monoclonal antibody auristatin con-jugates for cancer therapy. Nat Biotechnol 2003;21:778–84.

23. Francisco JA,CervenyCG,MeyerDL,MixanBJ, KlussmanK,ChaceDF, et al.cAC10-vcMMAE, an anti-CD30-monomethyl auristatin E conjugate withpotent and selective antitumor activity. Blood 2003;102:1458–65.

24. Chen Y, Clark S, Wong T, Dennis MS, Luis E, Zhong F, et al. Armedantibodies targeting the mucin repeats of the ovarian cancer antigen,MUC16, are highly efficacious in animal tumor models. Cancer Res2007;67:4924–32.

25. Boswell CA, Mundo EE, Zhang C, Bumbaca D, Valle NR, Kozak KR, et al.Impact of drug conjugation onpharmacokinetics and tissue distribution ofanti-STEAP1 antibody-drug conjugates in rats. Bioconjug Chem 1994;22:1994–2004.






26. Isola VJ, Eisenberg RJ, Siebert GR,HeilmanCJ,WilcoxWC,CohenGH. Finemapping of antigenic site II of herpes simplex virus glycoprotein D. J Virol1989;63:2325–34.

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

28. Institute of Laboratory Animal Resources. Guide for the care anduse of laboratory animals. Washington, DC: National AcademyPress; 1996.

29. Okeley NM, Miyamoto JB, Zhang X, Sanderson RJ, Benjamin DR, SieversEL, et al. Intracellular activation of SGN-35, a potent anti-CD30 antibody-drug conjugate. Clin Cancer Res 2010;16:888–97.

30. Senter PD. Potent antibody drug conjugates for cancer therapy. Curr OpinChem Biol 2009;13:235–44.

31. Burris HA, Gordon MS, Gerber DE, Spigel DR, Mendelson DS, Schiller JH,et al. A phase I study of DNIB0600A, an antibody-drug conjugate (ADC)targeting NaPi2b, in patients (pts) with non-small cell lung cancer(NSCLC) or platinum-resistant ovarian cancer (OC) 2014. J Clin Oncol32:5s, 2014 (suppl; abstr 2504).


Lin et al.




2015;21:5139-5150. Published OnlineFirst July 8, 2015.Clin Cancer Res Kedan Lin, Bonnee Rubinfeld, Crystal Zhang, et al. Ovarian Cancers

Small Cell Lung and−Drug Conjugate as a Therapeutic for Non−) AntibodySLC34A2Preclinical Development of an Anti-NaPi2b (

Updated version

10.1158/1078-0432.CCR-14-3383doi:

Access the most recent version of this article at:

Cited articles

http://clincancerres.aacrjournals.org/content/21/22/5139.full#ref-list-1

This article cites 29 articles, 14 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/21/22/5139.full#related-urls

This article has been cited by 3 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://clincancerres.aacrjournals.org/content/21/22/5139To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-14-3383

http://clincancerres.aacrjournals.org/content/21/22/5139.full#ref-list-1

http://clincancerres.aacrjournals.org/content/21/22/5139.full#related-urls

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

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/21/22/5139


full text (pdf) - clinical cancer...

Documents