effects of anti-vegf on pharmacokinetics, biodistribution...

Preclinical Development

Effects of Anti-VEGF on Pharmacokinetics, Biodistribution,and Tumor Penetration of Trastuzumab in a PreclinicalBreast Cancer Model

Cinthia V. Pastuskovas1, EduardoE.Mundo1, SimonP.Williams1, TapanK.Nayak2, JasonHo1,SheilaUlufatu1,Suzanna Clark1, Sarajane Ross1, Eric Cheng1, Kathryn Parsons-Reponte1, Gary Cain1, Marjie Van Hoy1,Nicholas Majidy1, Sheila Bheddah1, Josefa dela Cruz Chuh1, Katherine R. Kozak1, Nicholas Lewin-Koh1,Peter Nauka1, Daniela Bumbaca1, Mark Sliwkowski1, Jay Tibbitts1, Frank-Peter Theil1, Paul J. Fielder1,Leslie A. Khawli1, and C. Andrew Boswell1

AbstractBoth human epidermal growth factor receptor 2 (HER-2/neu) and VEGF overexpression correlate with

aggressive phenotypes and decreased survival among breast cancer patients. Concordantly, the combination

of trastuzumab (anti-HER2) with bevacizumab (anti-VEGF) has shown promising results in preclinical

xenograft studies and in clinical trials. However, despite the known antiangiogenic mechanism of anti-VEGF

antibodies, relatively little is known about their effects on the pharmacokinetics and tissue distribution of other

antibodies. This study aimed to measure the disposition properties, with a particular emphasis on tumor

uptake, of trastuzumab in the presence or absence of anti-VEGF. Radiolabeled trastuzumabwas administered

alone or in combination with an anti-VEGF antibody to mice bearing HER2-expressing KPL-4 breast cancer

xenografts. Biodistribution, autoradiography, and single-photon emission computed tomography–X-ray

computed tomography imaging all showed that anti-VEGF administration reduced accumulation of trastu-

zumab in tumors despite comparable blood exposures and similar distributions in most other tissues. A

similar trend was also observed for an isotype-matched IgG with no affinity for HER2, showing reduced

vascular permeability to macromolecules. Reduced tumor blood flow (P < 0.05) was observed following anti-

VEGF treatment, with no significant differences in the other physiologic parameters measured despite

immunohistochemical evidence of reduced vascular density. In conclusion, anti-VEGF preadministration

decreased tumor uptake of trastuzumab, and this phenomenon was mechanistically attributed to reduced

vascular permeability and blood perfusion. These findings may ultimately help inform dosing strategies to

achieve improved clinical outcomes. Mol Cancer Ther; 11(3); 752–62. �2012 AACR.

IntroductionDespite advances in prevention, diagnosis, and treat-

ment, cancer remains amajor health challenge. In the pastdecade, significant progress has been made in the field ofmonoclonal antibody (mAb) therapy (1). However, thestandard treatment option of single-drug therapy yields

limited successdue to lowrates of complete remission andresistance. There is a growing consensus that the future ofcancer treatment for solid tumors with immunotherapylies in a combination therapy approach (1, 2).

Trastuzumab is a humanized mAb that specificallytargets the extracellular domain of the human epidermalgrowth factor receptor-2 (HER2; refs. 3–5). TheHER2 geneis amplified or the receptor overexpressed in approxi-mately 15% to 25% of breast cancers (HER2-positivetumors; refs. 6, 7) and is associated with poor prognosis(8). Despite the success of trastuzumab treatment as asingle agent (9) or in combinationwith chemotherapy (10),intrinsic and acquired resistance to trastuzumab treat-ment has led to the investigation of trastuzumab in com-bination with other therapeutic agents (11).

In breast cancer, HER2 and VEGF signaling pathwaysare linked, as upregulation of VEGF occurs in HER2-over-expressing breast cancer in the most aggressive cases (12).VEGF is responsible for recruiting de novo vascularizationof the tumor, a process that is critical in all stages of

Authors' Affiliations: 1Genentech Research and Early Development,South San Francisco, California; and 2Pharma Research and Early Devel-opment, Hoffmann-La Roche Ltd., Basel, Switzerland

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

Corresponding Authors: Leslie A. Khawli, Early Development Pharma-cokinetics and Pharmacodynamics, Genentech, Inc., South San Fran-cisco, CA 94080. Phone: 650-225-6509; Fax: 650-742-5234; E-mail:[email protected]; and C. Andrew Boswell, Phone: 650-467-4603; Fax: 650-742-5234; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0742-T

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 11(3) March 2012752

on June 29, 2019. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst January 5, 2012; DOI: 10.1158/1535-7163.MCT-11-0742-T

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tumorigenesis (13–15). Consistently, bevacizumab (Avas-tin), a mAb specific for VEGFA (also referred to as VEGF)has shown efficacy against a variety of solid tumors (16).Anti-VEGF therapy has been evaluated in combinationwith chemotherapy in several tumor types, includingmetastatic breast cancer (17). A positive association wasshown between HER-2/neu and VEGF expression (18),with HER2 expression levels linked to therapeutic re-sponse (19). Thus, combining antibodies specific for HER2and VEGF could result in improved patient outcomecompared with either agent alone. Indeed, the combina-tion of bevacizumab and trastuzumab has shown partialtumor regression in xenograft models (20, 21). In a phase IIstudy, bevacizumab combined with trastuzumab improv-ed outcome in breast cancer patients with an overall re-sponse rate of 48% (22). Other promising strategies tofacilitate antitumor activity include simultaneous target-ing of HER2 and VEGF using a bi-specific antibody (23) orcombining trastuzumabwith an anti-VEGF fusion protein(24). At least 20 studies have been registered examiningbevacizumab plus trastuzumabwith cytotoxic chemother-apy and hormonal therapy in patientswithHER2-positivebreast cancer in neoadjuvant, adjuvant, and metastaticsettings (ClinicalTrials.gov; accessed on June 30 2011).In xenograft models of human tumor growth, both

human and mouse VEGF contribute to tumor angiogen-esis. Trastuzumab alone has been shown to possess anti-angiogenic properties through the downregulation oftumor produced VEGF (25, 26). Downregulation of VEGFsignaling in tumors is pursuedas anopportunity to restorevasculature to normalcy, a state requiring both spatial andtemporal control of VEGF levels (27). However, it is notclear what effect suppressing tumor vascularization bybevacizumabwill have on the distribution of trastuzumabto the tumor in vivo and resultant efficacy. In xenograftmodels, anti-VEGF therapy has induced rapid structuralandmorphologic changes in tumors (28), including reduc-tions in both vascular permeability (29) and interstitialfluid pressure (30), but the cumulative effect of thesechanges on trastuzumab distribution to and within thetumor is not known. To address these questions, weevaluated the effect of a mouse-specific anti-VEGF anti-body (B20-4.1),which also cross-reactswithhumanVEGF,on the tumor distribution and uptake of trastuzumab in aHER2-positive KPL-4 tumor xenograft model. In micebearing human tumor xenografts, bevacizumab wouldonly block tumor-derived human VEGF, whereasB20-4.1 blocks both tumor (human) and stroma-derived(murine)VEGF (31, 32). Trastuzumabor its isotype control(HER2 nonbinding antibody) were labeled with both125I and 111In, allowing assessment of both real-time kinet-ics and cumulative tumor uptake, respectively (33).

Materials and MethodsReagents and cell linesTrastuzumab (Herceptin), anti-VEGF antibody (B20-

4.1; refs. 31, 32), and an anti-herpes simplex virus glyco-

protein D (anti-gD, designated henceforth as IgG) usedas an isotype control for trastuzumab were obtainedfrom Genentech, Inc.. The HER2-expressing (3þ) humanbreast cancer cell line KPL-4, obtained in 2006 fromDr. J. Kurebayashi (34), was used in most studies. Thecells were cultured in RPMI 1640 media plus 1% L-gluta-minewith 10% FBS. TheKPL-4 cell linewas authenticatedby short tandem repeat profiling (AMEL: X; CSF1PO:11,13; D13S317: 12; D16S539:12; D5S818: 11,13; D7S820:9,10; TH01: 9; TPOX: 8; vWA: 14,19) and by single-nucle-otide polymorphism genotyping. Both methods indicatethat KPL-4 cells are unique when compared with a data-base of more than 1,000 human cancer cell lines.

A secondmodel of human breast cancer, the transgenicline MMTV-HER2 Fo5, was also tested (35). The Fo5model is a transgenic mouse model in which the humanHER2 gene, under transcriptional regulation of themurinemammary tumor virus promoter (MMTV-HER2),is overexpressed in mammary epithelium. The overex-pression causes spontaneous development of mammarytumors that overexpress the human HER2 receptor (35).The mammary tumor of one of these founder animals[founder #5 (Fo5)] has been propagated in subsequentgenerations of FVB mice by serial transplantation oftumor fragments. The model has been authenticatedas HER2 overexpressing (2�3þ) by HercepTest and byimmunohistochemistry.

RadiochemistryIodine-125 was obtained as sodium iodide in 10�5

N sodium hydroxide from Perkin Elmer. Trastuzu-mab (75 mg) was labeled randomly through tyrosineresidues at a specific activity of 11.3 mCi/mg with iodine-125 [125I] using the Iodogen method (Pierce Chemi-cal Co.; ref. 36). Radiosynthesis of 111In-trastuzumab(13.5 mCi/mg) was achieved through incubation of111InCl3 and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated (randomly throughlysines) mAb in 0.3 mol/L ammonium acetate pH 7at 37�C for 1 hour. Purification of all radioimmuno-conjugates was achieved using NAP5 columns equili-brated in PBS and confirmed by size-exclusion chro-matography. In addition to radiopurity and specificactivity, immunoreactivities of the final radiolabeledproducts were tested (see below).

ELISAELISA allowed comparison of 125I- or DOTA-conjugat-

ed mAbs with the unmodified mAbs. Briefly, a fixedconcentration of biotinylated trastuzumab (0.3 ng/mL)was mixed 1:1 with trastuzumab-DOTA or 125I-trastuzu-mab at varying concentrations (3 pg/mL-100 mg/mL) andcaptured on HER2 ECD (Genentech, Inc.) coated plates.Plates were developed using streptavidin–horseradishperoxidase (Amersham Pharmacia Biotech) and tetra-methyl benzidine substrate (Moss, Inc.). Kaleidographversion 4.03 (Synergy Software) was used to calculateIC50 values. For

125I-trastuzumab, a previously reported

Anti-VEGF Effects on Trastuzumab Disposition

www.aacrjournals.org Mol Cancer Ther; 11(3) March 2012 753




total trastuzumab direct binding ELISA was used (37).ELISA of 125I- or DOTA-conjugated antibodies showedmore than 80% recovery relative to unmodifiedantibodies.

Tissue distribution and pharmacokinetic studiesAll animal studies were conducted in accordance

with the guidelines of the American Association forAccreditation of Laboratory Animal Care and the Gen-entech Institutional Animal Care and Use Committee.C.B-17 Icr SCID (severe combined immunodeficient;Inbred) female mice (Charles River Laboratories),weighing between 20 to 25 g were inoculated in theright mammary fat pad with approximately 3 millionKPL-4 cells in a 50:50 suspension of Hanks’ BufferedSalt Solution (Invitrogen) and Matrigel (BD Biosciences)in at most 0.2 mL/mouse. When mean tumor volumereached 200 to 300 mm3, mice received a single bolusintravenous injection via the tail vein containing 111In-trastuzumab (10 mCi) and 125I-trastuzumab (5 mCi),along with 0.1, 1.4, and 17 mg/kg unlabeled antibody.The rationale for carrying out pharmacokinetic andtissue distribution studies at 3 vastly different doseswas not necessarily intended to mimic clinical exposure;instead, the goal was to study the biologic effects of anti-VEGF on trastuzumab uptake over a wide range oftumor receptor occupancies. To prevent thyroid seques-tration of 125I, 100 mL of 30 mg/mL of sodium iodidewas intraperitoneally administered 1 and 24 hoursbefore dosing. A control group for trastuzumab wasdosed with the mixture of 111In-IgG (10 mCi) and125I-IgG (5 mCi) with the addition of unlabeled IgG tocomplete a total dose of 18 mg/kg. Select mice receivedanti-VEGF at a dose of 10 mg/kg 24 hours before dosingradiolabeled trastuzumab or its control mAb. The doseof anti-VEGF mAb was based on previously reportedxenograft growth inhibition activity (16, 38), whereasthe time interval was based on reported statisticallysignificant reductions in vascular density of humanxenografts in mice at 24 hours following anti-VEGFadministration (28). Furthermore, a pharmacokineticmodel simulation indicated that either a 5 mg/kgtwice a week or 10 mg/kg weekly dosing regimenwould result in a minimum trough concentration atsteady state of 30 mg/mL, similar to that achieved inmore than 90% of bevacizumab patients (38).

Terminal tissue harvest was done at 24, 48, 120, and168 hours postinjection of trastuzumab or its controlIgG (n ¼ 3 per time point), and blood was collected viacardiac puncture under general anesthesia into lithiumheparin Microtainers (BD Biosciences). Tissues collectedconsisted of heart, right kidney, lungs, spleen, muscle(gastrocnemius), normal mammary fat pad, and tumor.Tissue handling and analysis provided counts perminutevalues, which were used to calculate the percent ofinjected dose per gram of tissue (%ID/g) and area underthe curve (AUC) as previously described (39–41). Notethat %ID/g is a dose-normalized unit of concentration.

Because animals were serially sacrificed with multipleblood samplings before takedown, we used a previouslyreported method (42) to calculate AUC0–7 and the differ-ence in AUC between groups with and without anti-VEGF treatment at each concentration, within eachorgan. P values were corrected for multiplicity withineach organ using a step down procedure (43).

Distribution of 111In-trastuzumab (17 mg/kg), with orwithout anti-VEGF pretreatment, was also assessed inmice bearing MMTV-HER2 Fo5 tumors (200–250 mm3)transplanted into themammary fat pad of Nu/Nu (nude-CRL) mice (Charles River Laboratories). Methods wereanalogous to those used in KPL-4 studies (see above).

SPECT-CT imagingIn vivo distribution was obtained by single–photon

emission computed tomography/X-ray computed tomo-graphy (SPECT-CT) using modification of previously re-ported methods (39, 40). Mice (n¼ 1) received an averagedose of 405 mCi (range: 383–416 mCi) of 111In-trastuzumabwith or without anti-VEGF pretreatment as describedabove. Immediately after CT acquisition, SPECT imageswere acquired on two 20% windows centered at the 173-and 247-keV photopeaks of 111In using a high-resolution5-pinhole collimator and a 5.5 cm radius of rotation.Mice were subsequently euthanized under sedation andtissues collected for gamma counting, in a manner iden-tical to that used for the nonimaging arm of the study.SPECT quantitation was accomplished using Amira soft-ware (TGS).

Autoradiographic imagingKPL4 tumor–bearing mice (n ¼ 3) with and without

anti-VEGF (as described above) were assessed by quan-titative whole-body autoradiography. Each mousereceived a single intravenous dose of 111In-trastuzumabplus unlabeled trastuzumab for a total dose of 17 mg/kg,100 mCi/mouse. Animals were processed for whole-body cryosectioning, exposed and imaged as describedpreviously (41).

Mechanistic physiologic studiesThe vascular volumes, interstitial volumes, and rates

of blood flow of KPL-4 tumors were measured in vivoby the indirect method for 99mTc red blood cell labeling(40), jugular cannula infusion of 111In-pentetate, and intra-venous bolus injection of 86Rb chloride, respectively, aspreviously described (39).

ImmunohistochemistryStaining was done on 4-mm thick formalin-fixed paraf-

fin-embedded tissue sections mounted on glass slides.Primary antibodies used were donkey antihuman IgG–biotin (Jackson ImmunoResearch), rabbit anti-panen-dothelial cell marker (clone Meca-32; Pharmingen), andrabbit anti-c-erbB-2 (HER-2; Dako). Detection was donewith Vectastain ABC Elite reagent (Vector Labs) andPierce metal enhanced DAB (Thermo Scientific).

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Mol Cancer Ther; 11(3) March 2012 Molecular Cancer Therapeutics754




ResultsRadionuclide comparisonThe blood profiles of both 125I and 111In isotopes were

similar throughout the study, declining progressivelyover time (Fig. 1). Tumor uptake of 111In-trastuzumabat 0.1 mg/kg gradually increased before peaking at38.7 � 12.5%ID/g at 120 hours postdose (Fig. 2A) andremained at 30.7 � 0.5%ID/g at 168 hours. Simi-larly, maximum tumor uptake of 125I-trastuzumab at0.1 mg/kg (12.9 � 8.0%ID/g) occurred 120 hours post-dose and gradually decreased with time to 7.0 �2.1%ID/g at 168 hours postinjection. Two- to 3-foldhigher uptake of 111In-trastuzumab compared with125I-trastuzumab was generally observed at most timepoints and doses due to residualization (i.e., tumor cellretention) of radiometal (Fig. 2). In contrast, uptake of111In-trastuzumab in most nonmalignant tissues wassimilar to that of 125I-trastuzumab at all doses (Fig. 3,Supplementary Table S1).

Antigen specificity and dose escalationThe dose-normalized concentration of radiolabeled

trastuzumab was similar in blood (Fig. 1) and normaltissues (Fig. 3) at all doses of trastuzumab (0.1, 1.4, and 17mg/kg). Similar levels were observed for radiolabeledIgGat 18mg/kg. Tumoruptake of 111In-trastuzumabat alldoses was higher than that of 111In-IgG at 17 to 18 mg/kg(Fig. 2).An inverse relationship was observed between trastu-

zumab dose and tumor uptake of 111In- and 125I-trastu-zumab (Fig. 3A and C). The highest tumor uptake was

Figure 1. Blood pharmacokinetic curves of 125I-labeled (A) and111In-labeled (B) antibodies at various doses through 7 days. Soliddata symbols correspond to control mice, whereas open symbolsindicate mice treated with anti-VEGF at 24 hours before traceradministration.

Figure 2. Tumor pharmacokineticcurves with and without anti-VEGF(B20.4.1) pretreatment: trastuzumabat doses of 0.1 mg/kg (A), 1.4 mg/kg(B), 17mg/kg (C), and a control IgG ata dose of 18 mg/kg (D).






observed at a dose of 0.1 to 1.4 mg/kg (38.7 � 12.5 to45.8 � 4.4%ID/g and 12.9 � 8.0 to 10.9 � 2.6%ID/g for111In- and 125I-trastuzumab, respectively).With the excep-tion of spleen and liver, normal tissue uptake was notaffected by trastuzumab dose. Uptake of 111In-IgG at18 mg/kg was low and nearly constant, ranging from7.2 � 2.0 to 8.4 � 0.5%ID/g in tumor compared with16.6 � 2.0 to 26.3 � 10.2%ID/g for uptake of 111In-trastu-zumab at 17 mg/kg (Fig. 2D). Relative to radiolabeledtrastuzumab, much lower 120-hour tumor uptakes ofonly 8.2 � 1.3 and 4.20 � 0.030%ID/g for 111In- and125I-IgG, respectively, were detected (Fig. 3A and C).

Dose-normalized cumulative tumor uptake of 111In-trastuzumab at 0.1 mg/kg was 204 � 5%ID/g � dand was higher than that of 125I-trastuzumab (68.9 �3.5%ID/g � d) (Supplementary Table S1). Dose escala-tion caused a significant decrease in measured AUC0–7

of radiolabeled trastuzumab, with a 25% to 30% lowerdose-normalized cumulative tumor uptake of 125I- and111In-trastuzumab, respectively, at 17 mg/kg (48.5 � 4.1and 153 � 5%ID/g � d) than at 0.1 mg/kg (68.9 � 3.5and 204 � 5%ID/g � d). In contrast, calculated AUC0–7

in the normal tissues showed approximately similarvalues at all doses.

Anti-VEGF pretreatmentAs shown in Fig. 2A, non-pretreatedmice at tracer dose

(0.1 mg/kg) had 120-hour tumor uptakes of 38.7 � 12.5and 12.9 � 8.0%ID/g for 111In- and 125I-trastuzumab,respectively, whereas mice pretreated with anti-VEGFhad 120-hour tumor uptakes of 18.0 � 5.8 and 6.0 �

4.0%ID/g, respectively. Similarly, mice injected with anescalating dose of trastuzumab had reduced tumor accu-mulation at all time points when pretreated with anti-VEGF (Fig. 2A–C), whereas radioactivity levels weresimilar in normal tissues (Fig. 3). Accumulation of 111In-and 125I-IgG in tumor was reduced after pretreatmentwith anti-VEGF, as shown by the lower uptake that wasachieved with the 18 mg/kg dose of IgG (Fig. 2D), where-as the uptake was similar in normal tissues (Fig. 3).

Anti-VEGF pretreatment resulted in lower tumoraccumulation of tracer trastuzumab (0.1 mg/kg) relativeto the non-pretreated group (204 vs. 95%ID/g � dfor 111In-trastuzumab, and 70 vs. 33%ID/g � d for125I-trastuzumab; Fig. 4, Supplementary Table S1). Simi-larly, with higher doses (1.4 and 17 mg/kg), the magni-tude of tumor AUC0–7 diminishes, as previously seen attracer dose, without altering the biodistribution of anti-body in blood and normal tissues. Consistent with datafrom the KPL-4 tumor model herein, anti-VEGF pre- orposttreatment also reduced overall trastuzumab uptakeinto MMTV-HER2 Fo5 tumors (Supplementary Fig. S1).

SPECT-CT imagingForty-eight-hour SPECT-CT images of 111In-trastuzu-

mab in KPL-4 tumor-bearing mice showed higher radio-tracer uptake in tumor with tracer antibody dose(1.4 mg/kg; Fig. 5A) compared with tumor uptake inanimals receiving 14 mg/kg (Fig. 5B). Analogous to thenonimaging biodistribution results shown in Fig. 3A, ahigh codose of nonradioactive trastuzumab causeda reduction in tumor uptake of 111In-trastuzumab by

Figure 3. Tissue distribution of111In-labeled (A and B) and125I-labeled (C and D) antibodies at120-hour postinjection. Data in Band D are derived from micereceiving anti-VEGF at 24 hoursbefore tracer injection.

Pastuskovas et al.





SPECT-CT at 48 hours postinjection (31.5% and 18.5%in trastuzumab-only and anti-VEGF–treated mice,respectively) and showed evidence of specific accumu-lation of trastuzumab in tumors.Forty-eight-hour SPECT-CT images of 111In-trastuzu-

mab in KPL-4 tumor–bearing mice with anti-VEGF pre-treatment showed a 58.2% and 50.2% reduction in tumors,at total trastuzumab doses of 1.4 mg/kg and 14 mg/kg,respectively, compared with animals receiving no anti-VEGF (Fig. 5A and B). Similar percent reductions(50%–60%) in tumor uptake were observed by SPECT-CTat 72 hours and showed excellent agreement (49%–51%)with corroborative terminal tissue distribution done bygamma counting immediately after noninvasive imaging(data not shown). Furthermore, these anti-VEGF–medi-ated reductions in tumor uptake are fairly consistentwith the nonimaging biodistribution results shownin Fig. 2A and C, where at 48 hours, reductions in111In-trastuzumab uptake of 61% and 56% were observedat 0.1 and 17 mg/kg, respectively.

Autoradiographic imagingIntratumoral localization of 111In-trastuzumab inKPL-4

tumor–bearing mice, as determined by quantitative auto-

radiography, is depicted in Fig. 5E and F. The left panelshows representative digital pictures of sagittal tumorsections 48 hours after administration of 111In-trastuzu-mab at 17 mg/kg, with and without anti-VEGF pretreat-ment. Autoradiographic imaging of these sections, rightpanel, showed a differential spatial distribution of111In-trastuzumab as a result of anti-VEGF pretreatment.A more homogenous distribution of 111In-trastuzumab isobserved in tumors receiving 111In-trastuzumab alonecompared with that of anti-VEGF–pretreated tumors. Inaddition, a 44 � 5% reduction of trastuzumab obtainedfrom the autoradiogram analysis at the 48-hour timepoint is in agreement with the results obtained fromSPECT-CT quantification and direct biodistribution mea-surements of the tumors.

Mechanistic physiologic studiesThemeasured physiologic parameters for KPL-4 tumor

and various tissues in the presence and absence of anti-VEGF are summarized in Supplementary Table S2.Among the 3 parameters measured, only blood flow(Q) in tumor showed a statistically significant differencebetween anti-VEGF treatment and lack thereof. Further-more, no differences were detected in any nonmalignant

Figure 4. Absolute difference inAUC (%ID/g � d), relative to control,due to the effect of a 24-hourpretreatment of the anti-VEGFantibody, B20-4.1.






tissue. The average rates of blood flow calculated forKPL-4 xenografts without (99 � 28 mL/g/min) and with(49 � 12 mL/g/min) anti-VEGF pretreatment were statis-tically different (P < 0.05) (Supplementary Table S2).

ImmunohistochemistryDecreased vasculature was observed by MECA-32

staining in tumors treated with anti-VEGF and trastu-zumab compared with trastuzumab-only treatment(Fig. 6A and B). Remaining vasculature was presentprimarily at the periphery of tumor lobules. Boundtrastuzumab (Fig. 6E and F) was predominately limitedto the periphery of tumor lobules in anti-VEGF/trastu-zumab-treated tumors, consistent with distribution ofdetected vascular elements in these tumors. HER2antigen (Fig. 6C and D) was detected with slightly less

intensity in anti-VEGF and trastuzumab-treated groupscompared with trastuzumab-only–treated groups. Thedistribution of HER2 sites was variable in anti-VEGF/trastuzumab-treated tumors and not necessarily asso-ciated with peripheral vascular distribution.

DiscussionThese studies represent the first direct comparison of

trastuzumab disposition alone and in the presence of anti-VEGF therapy using a species cross-reactive anti-VEGFantibody (B20-4.1) in a xenograft tumor model in mice.The cross-reactive nature of the B20-4.1 antibody is ofimportance in that it displays immunoreactivity withboth human VEGF (tumor derived) and murine (host)VEGF (stromal/vasculature derived; ref. 32), yielding an

Figure 5. SPECT-CT (A–D) andautoradiograms with theirrespective pseudocolor heat map(E and F) imaging data acquired inmice following intravenousadministration of 111In-trastuzumab. For SPECT-CTimages, 3-dimensional volumerenderings from a coronalperspective (top), coronaltomographic slices (middle), andaxial tomographic slices (bottom)are displayed. Mice received111In-trastuzumab tracer only (Aand C) or were coinjected with adose of 14 to 17 mg/kg (B, D–F).Only mice in C, D, and F received asingle intravenous dose of anti-VEGF (10mg/kg) at 24hours beforetracer injection.

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experimental system that is somewhat analogous to theclinical scenario. This approach enabled quantitativeanalysis of trastuzumab distribution to be assessed withboth 125I- and 111In-radionuclides, in view of known dif-ferences in their biodistributionpatternswhen conjugatedto antibodies (33). This study also showed the effects ofpreadministration of anti-VEGF on biodistribution andpharmacokinetics of radiolabeled trastuzumab. Further-more, the results reaffirmed that suitably labeled trastu-zumab can be used for noninvasive detection of boundtrastuzumab in preclinical tumor models, with sufficientsensitivity to detect anti-VEGF–mediated changes intumor pharmacokinetics.Tumor uptakewas superior for 111In-trastuzumab at all

time points studied compared with that of 125I-trastuzu-mab. This is expected due to the residualizing propertiesof the 111In-DOTA label that is trapped intracellularlyafter internalization and proteolytic degradation of tar-geting antibody by tumor cells. Indium-111–trastuzumabaccumulates at the target site, enabling the cumulative

assessment of uptake over time (33). In contrast, trastu-zumab labeled with nonresidualizing 125I showed lowerretention of 125I-trastuzumab, allowing for the assessmentof real-time kinetics of antibody distribution (33).

The effect of anti-VEGF on tumor uptake of trastuzu-mab was evaluated by coadministering 111In-DOTA- and125I-labeled trastuzumab with or without pretreatment ofanti-VEGF. Irrespective of the trastuzumab radioactiveprobe or dose, there was no anti-VEGF effect on theoverall exposure of trastuzumab in the blood (Figs. 2and 4). These data show that any change in trastuzumabuptake by tumors in the presence of anti-VEGF was nota consequence of altered exposure of trastuzumab butwas rather a tumor-specific phenomenon.

Asdepicted inFig. 1, preadministrationwith anti-VEGFresulted in decreased tumor uptake of the anti-HER2antibody in the KPL-4 xenograft model and was notdependent on the dose of trastuzumab administeredor the radiolabeling probe used. This effect of anti-VEGFwas more pronounced when quantifying trastuzumab

Figure 6. Immunohistochemicalstaining of KPL-4 tumor sections forthe MECA-32 vascular endotheliummarker (brown;A,B),HER2 receptors(pink; C, D), and bound trastuzumab(blue; E, F). Images in A, C, andE correspond to trastuzumab(17 mg/kg) only treated mice,whereas those in B, D, and F arederived from mice receiving a singleanti-VEGF dose (10 mg/kg) 24 hoursbefore trastuzumab. Tumors wereharvested at 24 hours afterintravenous administration oftrastuzumab. Scale bar, 500 mm.Insets display same tumor sectionsat a larger scale (2.0 mm).

A B

C D

E F






accumulation by using 111In-DOTA. Furthermore, theeffect of anti-VEGF on 111In-trastuzumab tumor uptakewas less pronounced by day 7, possibly a consequenceof diminished effects of the anti-VEGF antibody due toclearance. To show that these results were not uniqueto trastuzumab, tracer studies were carried out to com-pare uptake using an isotype control IgG. Interestingly,anti-VEGF also diminished tumor uptake of the controlantibody (Fig. 1D). The decrease in tumor uptake of IgGconfirms that at least one mechanism of action for anti-VEGF is to produce vascular permeability changes in thetumor, whereas that of most normal organs was unaffect-ed (Figs. 3 and 4). This is an important observation, as itsuggests that anti-VEGF treatment has the potential toreduce antibody distribution to the tumor independentof antibody specificity. Reduced tumor uptake of radi-olabeled trastuzumab by anti-VEGF administered at24 hours before or after the tracer was also observed inan additional tumor model (Fo5; Supplementary Fig. S1),indicating that the effect of anti-VEGF on tumor uptakeof trastuzumab is independent of the dose schedule orhigh HER2-expressing tumor model used.

Although overall distribution to normal tissues waslargely unaffected by anti-VEGF, it must be noted that atlower doses (0.1 and 1.4 mg/kg), trastuzumab retentionin the spleen and liver was diminished in the presenceof anti-VEGF (compare Fig. 3A and B, and Fig. 3C and D).This is likely a consequence of nonlabeled antibody com-petition for binding to FcgR due to extremely low endog-enous IgG levels in SCID mice (44), as this effect wasnot seen when greater nonlabeled antibody concentra-tions were administered (17 mg/kg trastuzumab or10 mg/kg anti-VEGF). The kidney presented with a trendfor greater uptake of 111In-antibody (Fig. 3A and B, andSupplementary Table S1) despite treatment and isotype,which is consistent with the renal tubular reabsorptionof 111In-DOTA degradation products (45). In normal tis-sues, distribution of trastuzumab and an isotype controldid not distinguish from each other, again consistent withthe lack of HER2 expression in these tissues.

The acute effect of anti-VEGF on trastuzumab tumoruptake observed by the biodistribution study describedabove was confirmed by SPECT-CT. At a low dose of111In-trastuzumab, anti-VEGF administration 24 hoursbefore trastuzumab caused a 58.2% reduction in tumoruptake relative to untreated (Fig. 5A and C). At the14 mg/kg dose of 111In-trastuzumab, anti-VEGF ledto a 50.3% reduction in tumor uptake (Fig. 5B and D).Consistently, by autoradiography, a 42% reduction intumor uptake of a 17 mg/kg 111In-trastuzumab dose wasobserved in the presence of anti-VEGF (Fig. 5E and F).

Tumor bloodperfusion, asmeasured by radiorubidiumuptake and expressed as a function of cardiac output,had a baseline value of 98.9 � 28.0 mL/g/min in theabsence of anti-VEGF (Supplementary Table S2). How-ever, at 24 hours after treatment with anti-VEGF, the rateof blood flow had significantly (P < 0.05) decreased to48.5 � 12.1 mL/g/min (Supplementary Table S2). This

effectwas tumor specific, as anti-VEGFdid not alter bloodflow to nonmalignant tissues, and these findings agreewith our previous data (39). The rubidium radionuclide86Rbþ is an idealmarker for regional blood flow in tumors

and other tissues, because its small size (roughly 1.5 A�)

dictates that its entry into tissues in a brief (<1.5 min) timeperiod should be dominated by blood flow and thusminimally permeability limited (46). Such changes intumor blood flow dynamics may be related to nitricoxide (NO) levels (47), as inhibition of NO production isone of the documented consequences of VEGF inhibition(48). Indeed, our findings are consistent with a previousclinical study using bevacizumab, in which observedreductions in the volume transfer constant (Ktrans) bydynamic contrast-enhanced MRI (DCE-MRI) were con-sistent with decreased tumor blood flow and/or reduc-tion in vessel permeability (28). Ktrans is a compositeestimate of both the blood flow and the permeabilitysurface area product per unit volume of tissue for trans-endothelial transport between plasma and extravascularspace.

Although a reduction in relative blood volume oftumors following treatmentwith adifferent cross-reactiveanti-VEGF antibody was previously reported (28), weobserved no anti-VEGF–induced change in tumor bloodvolume (Supplementary Table S2). This could be relatedto the method of measurement, as blood volumes wereassessed by noninvasive DCE ultrasound imaging ofmicrobubbles (1–3 mm) in the previous work (28) and bydetection of radiolabeled murine red blood cells (6 mm)following invasive tumor harvest in thiswork.Despite theapparent discrepancy in anti-VEGF effects on tumorblood volume, reductions in tumor vascular density wereobserved by immunohistochemistry in both the previous(28) and current studies (Fig. 6A and B). These seeminglycontradictory findings raise the possibility that anti-VEGF may achieve tumor vessel normalization (27), inpart, by pruning nonfunctional blood vessels carryinglittle to no blood.

Anti-VEGF decreased vasculature density and limit-ed the distribution of trastuzumab to the periphery oftumor lobules. Representative tumor sections are shownin Fig. 6, revealing that anti-VEGF not only reducedtumor uptake of trastuzumab but also restricted itsdistribution to the periphery of the tumor (Fig. 6E andF). Decreased vascular staining in anti-VEGF pretreat-ed tumors is obvious at the periphery of neoplasticlobules, especially in the interior of mass comparedwith mice receiving trastuzumab only (Fig. 6A andB). It seems reasonable to speculate from these ex vivovisualizations that the relatively decreased tumor vas-culature may regulate, at least in part, the rate of macro-molecule extravasation across tumor blood vessels longknown to be associated with the qualitatively poorpenetration into the tumors (49). These results werecorroborated by both autoradiography and immuno-histochemical staining observed in Figs. 4F and 5F,respectively. Moderate to high expression of HER2

Pastuskovas et al.





was evenly distributed throughout the tumor mass inmice receiving trastuzumab-only (4þ) and anti-VEGFfollowed by trastuzumab (3þ; Fig. 6C and D), respec-tively. The slightly weaker staining of HER2 in anti-VEGF–treated tumors raises the possibility that down-regulation of HER2 expression by anti-VEGF may be atleast partially responsible for the decrease in trastuzu-mab tumor uptake.Despite the results herein, considerable preclinical

evidence for efficacy of trastuzumab in combinationwith bevacizumab must still be weighed (20, 21). Fur-thermore, a phase II trial evaluating combination ofbevacizumab and trastuzumab in first-line treatment of50 patients with HER2-positive advanced breast cancerreported encouraging results (50). Nevertheless, furtherinvestigations are necessary to better understand thecomplex interplay between each constituent of the com-bination and the resulting effects on overall patientbenefit.In conclusion, these results show that (i) tumor

uptake of trastuzumab decreased with concomitantadministration of an anti-VEGF antibody; (ii) bloodpharmacokinetics were not impacted by anti-VEGFadministration, indicating that the differences in tumoruptake were not blood exposure driven; (iii) the impactof an anti-VEGF antibody on the tumor uptake of apostadministered antibody was independent of anti-

body specificity and tumor type; (iv) the effect ofanti-VEGF was restricted to tumors as most normaltissues remained unaffected; and (v) mechanistic stud-ies suggest that reductions in both tumor blood flowand vascular permeability to macromolecules contrib-ute to the observed changes in tumor pharmacokineticsof antibodies.

Disclosure of Potential Conflicts of InterestAll authors have held financial interest as employees of Hoffmann-

La Roche or Genentech, a member of the Roche Group. Herceptin(trastuzumab) is marketed in the U.S. by Genentech and internationallyby Roche.

AcknowledgmentsThe authors thank RichardNeve for scientific discussions andMichelle

Schweiger, Jose Imperio, Kirsten Messick, Nicole Valle, Cynthia Young,Nina Ljumanovic, Bernadette Johnstone, and Shannon Stainton for excel-lent animal study support and guidance.

Grant SupportAll financial support was provided by Genentech, a member of the

Roche Group.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 September 19, 2011; revised December 1, 2011; acceptedDecember 21, 2011; published OnlineFirst January 5, 2012.

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2012;11:752-762. Published OnlineFirst January 5, 2012.Mol Cancer Ther Cinthia V. Pastuskovas, Eduardo E. Mundo, Simon P. Williams, et al. ModelTumor Penetration of Trastuzumab in a Preclinical Breast Cancer Effects of Anti-VEGF on Pharmacokinetics, Biodistribution, and

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