Intrathecal Viral Vector Delivery of ... - Cancer ResearchWilliam T. Rothwell1, Peter Bell1, Laura K. Richman1, Maria P. Limberis1, Anna P.Tretiakova1, Mingyao Li1,2, and James M.Wilson1

Tumor Biology and Immunology

Intrathecal Viral Vector Delivery of TrastuzumabPrevents or Inhibits Tumor Growth of HumanHER2-Positive Xenografts in MiceWilliam T. Rothwell1, Peter Bell1, Laura K. Richman1, Maria P. Limberis1, Anna P.Tretiakova1,Mingyao Li1,2, and James M.Wilson1

Abstract

Breast cancer brain metastases are a deadly sequela ofprimary breast tumors that overexpress human epidermalgrowth factor receptor 2 (HER2); median survival for patientswith these tumors is 10 to 13 months from the time ofdiagnosis. Current treatments for HER2-positive breast cancerbrain metastases are invasive, toxic, and largely ineffective.Here, we have developed an adeno-associated virus serotype 9(AAV9) vector to express the anti-HER2monoclonal antibodytrastuzumab (Herceptin) in vivo. A single prophylactic intra-thecal administration of AAV9.trastuzumab vector in a novelorthotopic Rag1�/�murine xenograft model of HER2-positivebreast cancer brain metastases significantly increased mediansurvival, attenuated brain tumor growth, and preserved both

the HER2 antigen specificity and the natural killer cell–asso-ciated mechanism of action of trastuzumab. When adminis-tered as a tumor treatment, AAV9.trastuzumab increasedmedian survival. Dose-escalation studies revealed that higherdoses of AAV9.trastuzumab resulted in smaller tumorvolumes. Our results indicate that intrathecal AAV9.trastuzu-mab may provide significant antitumor activity in patientswith HER2-positive breast cancer brain metastases.

Significance: Intrathecal delivery of trastuzumab via adeno-associated virus has the potential to become a novel, integralpart of adjuvant therapy for patients with HER2-positive breastcancer brain metastases. Cancer Res; 78(21); 6171–82.�2018 AACR.

IntroductionBreast cancer is the most commonly diagnosed malignancy in

women in the United States with an estimated 268,670 new casesin 2018 (1). Approximately 20% of breast cancers overexpresshuman epidermal growth factor receptor 2 (HER2) and areconsidered to be more aggressive and more likely to metastasizeto the brain than other breast cancer subtypes (2–4). About 30%of patients with metastatic HER2-positive (HER2þ) breast cancerwill develop breast cancer brain metastases (BCBM; refs. 5–8). Inthe registHER prospective study of patients with newly diagnosedHER2þ metastatic breast cancer, 37.3% of the 1,012 patientsstudied developed brain metastases within 10.8 months of theinitial diagnosis of metastatic disease (9). Brain metastases cansignificantly lower patient quality of life by inducing nausea,sensory loss, aphasia, motor deficits, ataxia, seizures, stroke, andparalysis (3, 10–12). The median age at diagnosis of HER2þ

BCBM is 48 years (13), and the incidence of HER2þ BCBM isrising (4). Evidence suggests that this increase is due to many

factors. Targeted, effective therapies for HER2þ tumors in theperiphery have increased the duration of patient survival, therebyallowing adequate time for outgrowth of brain metastases thatwould have otherwise remained subclinical before death (10, 14).Importantly, many of these therapies, including biological ther-apeutics, do not reach adequate concentrations in cerebrospinalfluid (CSF) after systemic administration (15, 16).

The current standard-of-care treatments for HER2þ BCBM areinvasive, can cause cognitive impairment, and provide subopti-mal survival benefits. Patients often undergo a combination ofneurosurgical tumor resection, stereotaxic radiosurgery, whole-brain radiotherapy, systemic and/or intrathecal (i.t.) chemother-apy, steroids, or anti-HER2 agents (3, 10, 14). Even with thesetreatments, survival from the time of diagnosis of HER2þ BCBMranges from 3 to 25 months with a median of 10 to 13 months(5, 9, 17, 18). Clearly, there is an unmet need for more effective,targeted treatments for patients with HER2þ BCBM.

Trastuzumab (Herceptin, Roche) is a humanized monoclonalantibody (mAb) directed against HER2 that extends survival ofpatients when used with chemotherapy to treat primary andsystemically metastatic HER2þ disease (5, 19, 20). However,trastuzumab does not cross the intact blood–brain barrier to treatcentral nervous system (CNS) tumors (2, 3). Additionally, theCSFconcentration of trastuzumab after intravenous (i.v.) administra-tion is 300- to 400-fold lower than that in serum (15, 16). As such,patients with concurrent HER2þ BCBM and systemic HER2þ

disease who receive i.v. trastuzumab often experience regressionor stabilization of systemic tumor burden while brain metastasesprogress (5).

Trastuzumab administered i.t. has been reported to increasesurvival and delay progression of HER2þ brain metastases. Bous-quet and colleagues administered i.t. trastuzumab to a patient

1Gene Therapy Program, Department of Medicine, Perelman School of Medicine,University of Pennsylvania, Philadelphia, Pennsylvania. 2Department of Biosta-tistics and Epidemiology, Perelman School of Medicine, University of Pennsyl-vania, Philadelphia, Pennsylvania.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: James M.Wilson, University of Pennsylvania, 125 S. 31stSt, TRL 1200, Philadelphia, PA 19104. Phone: 215-573-9020; Fax: 215-494-5444;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-18-0363

�2018 American Association for Cancer Research.

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with HER2þ cerebellar and epidural breast cancer metastases,resulting in a 6-month halt in disease progression (21). Colozzaand colleagues described a patient with HER2þ intraparenchymalcortical metastases who was treated for 19 months with i.t.trastuzumab (22). In both case reports, patients were still aliveat the time of publication. A 2013 meta-analysis by Zagouri andcolleagues demonstrated that themedian survival of patientswithHER2þ leptomeningeal carcinomatosis, a particularly deadlyform of BCBM, increases from 5.9 months in historical controlsto 13.5 months with i.t. trastuzumab treatment (23). A recentphase I clinical trial in Europe demonstrated that administeringdoses of up to 150 mg of trastuzumab i.t. to patients withleptomeningealHER2þ resulted inno serious adverse events (24).

Despite these promising reports, i.t. administration of trastu-zumab has disadvantages. For instance, multiple i.t. administra-tions are required. Perhaps more importantly, the normal, rapidturnover of CSF results in a widely fluctuating pharmacokineticprofile of trastuzumab inCSF, resulting in aCSFhalf-life of just 12hours (21, 25). It is therefore unlikely that tumor cells in the CNSreceive optimal exposure to trastuzumab after i.t. administration.

Gene therapy offers the potential for a one-shot solution to theproblem of mAb delivery across the blood–brain barrier. Adeno-associated viral (AAV) vectors, particularly serotype 9, can safelyand efficiently deliver exogenous genes, such as the gene fortrastuzumab, to neurons and astrocytes throughout the brain andspinal cord after a single i.t. administration. This results in long-term, stable expression of the transgene product in the brainparenchyma and CSF (26, 27).

In this study, we aimed to use AAV9.trastuzumab delivered i.t.to bypass the blood–brain barrier for localized expression oftrastuzumab in situ. We developed a novel Rag1�/� murineorthotopic xenograft model of HER2þ BCBM, and then deliveredAAV9.trastuzumab i.t. by intracranioventricular (ICV) injectioneither as tumor prophylaxis or treatment. In both cases, a singledose of i.t. AAV9.trastuzumab significantly extended mediansurvival of mice compared with no treatment or control AAVvector treatment. Higher doses of vector led to smaller tumorvolumes when measured 35 days after tumor implantation. Wealso showed that trastuzumab expressed byCNS cells still binds toHER2 on tumors and maintains the clinical product's principalmechanismof action against tumors: facilitating antibody-depen-dent cell-mediated cytotoxicity through natural killer (NK) cells.Looking ahead, we predict that i.t. AAV9.trastuzumab couldprolong survival of patients with existing HER2þ CNSmetastasesin addition to patients with primary or metastatic HER2þ breastcancer at risk for developing BCBM.

Materials and MethodsExperimental design

Six- to 9-week-old B6.129S7-Rag1tm1Mom/J (Rag1�/�) andNOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were obtained fromThe Jackson Laboratory and housed at the barrier facility of theTranslational Research Laboratories Vivarium at the University ofPennsylvania. A female cynomolgus macaque was obtained fromCovance Inc. and housed at the animal facility of the BiomedicalResearch Building II/III at the University of Pennsylvania. Allanimals were maintained according to NIH guidelines for thecare and use of animals in research.

All procedures and protocols were approved by the Institution-al Animal Care and Use Committee of the University of Penn-

sylvania. The 201Ig IA (201IA) controlmAb is a rhesus antisimianimmunodeficiency virus IgG immunoadhesin. The 2.10A controlmAb is a rhesus antisimian/human immunodeficiency virus IgG.Vectors expressing these two antibodies served as negative con-trols for vector-mediated antibody production. AAV9.null, whichserves as a negative control for the presence of vector transductionin the CNS, has an intact genome and expression cassette but hasno transgene. Phosphate-buffered saline (PBS) was used as acontrol for the volume of vector administration. AAV9var is aclosely related AAV9 variant that performs similarly in vivo.

We administered vector to mice at least 21 days before tumorimplantation in the prophylaxis studies and 3 days after tumorimplantation in the treatment studies. Prophylactic AAV9var.trastuzumab was administered 2 weeks before tumor implanta-tion. Ten to 14 days are required for vector expression to peak andreach steady state; we wanted to ensure expression was at a steadystate when tumor was injected.

We used aminimumof 8mice per experimental group to allowfor robust statistical analysis in case mice were euthanized due tocomplications during or after tumor implantation. The exceptionis the pilot study represented in Supplementary Fig. S1A and S1B.The large prophylaxis study began with 20 mice per group, thetreatment study with 8 mice per group, and the NSG and NK celldepletion studies with 12 mice per group. Macrophage depletionstudies began with 9 mice per group, with an additional 2 miceadded to AAV9.trastuzumab–treated arms due to clodronate-related toxicity. Mice found dead rather than euthanized wereincluded only in the survival analyses and not in downstreamtransgene expression, histologic, or biodistribution analysis.Studies were conducted without blinding.

Statistical analysisSurvival study and tumor volume P values were calculated

using the log-rank (Mantel–Cox) test in GraphPad Prism softwarefor Windows 7. Hazard ratios were calculated with a Cox regres-sion model using the sts package (StataCorp).

Vector constructionSequences matching the World Health Organization–pub-

lished amino acid sequences of the heavy and light chains oftrastuzumab were back translated, codon optimized, and synthe-sized by GeneArt (Life Technologies). Heavy and light chainsequences were preceded by a human IL2 secretion signal. Theheavy and light chain sequences were cloned into an AAV expres-sion construct containing an upstream hybrid cytomegalovirus(CMV) immediate early enhancer/chicken b-actin promoter, achimeric intron (Promega), and a downstream simian virus40 (SV40) polyadenylation signal. The heavy and light chainsequences were separated from each other by a foot-and-mouthdisease virus-derived self-cleaving peptide to ensure 1:1 produc-tion of heavy and light chainprotein. The constructwasflankedbyAAV2 inverted terminal repeats. The resulting pAAV.CMV.PI.tras-tuzumab.SV40 expression construct was packaged in an AAV9or AAV9var capsid by triple transfection of 293 cells and purifiedas previously described (28). The regulatory elements in theAAV9.201IA expression cassette are a chicken b-actin (CB7)promoter, a chimeric intron, and a downstream rabbit beta-globin polyadenylation signal (rGB). The regulatory elements inthe AAV9.null expression cassette are a thyroid hormone-bindingglobulin promoter, amicroglobin/bikunin enhancer (TBG), and abovine growth hormone (bGH) polyadenylation signal. Vector

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was titrated using standard quantitative polymerase chain reac-tion (qPCR) or digital droplet PCR (AAV9var.trastuzumab only).AAV9.CMV.PI.2.10AmAb.SV40, AAV9.CB7.PI.201IA.rBG, andAAV9.TBG.PI.null.bGH were obtained from the Penn VectorCore.

Vector administrationFor tumor prophylaxis, mice were injected ICV with 1 � 1011

genome copies (GC) of AAV9.CMV.PI.trastuzumab.SV40, AAV9.CB7.PI.201IA.rBG, AAV9.TBG.PI.null.bGH, or AAV9.CMV.PI.2.10AmAb.SV40 diluted in sterile PBS at least 21 days priorto BT474.M1.ffluc tumor implantation. For tumor treatment,mice received BT474.M1.ffluc tumors first, and vector was admin-istered by ICV injection 3 days after tumor implantation. AAV9-var.CMV.PI.trastuzumab.SV40 was administered in half-logsfrom 1e10 to 3e11 GC/mouse for tumor volume studies.

Orthotopic xenograft model of HER2þ breast cancer braintumors in Rag1�/� and NSG mice

The HER2þ BT474.M1 human ductal carcinoma cell line was agenerous gift from Lewis Chodosh and Jason Ruth (University ofPennsylvania). IMPACT I testing (IDEXX BioResearch) indicatedno Mycoplasma spp. contamination. Cell authentication was notconducted. Cells were transduced with the lentiviral vector VSVG.HIV.SIN.cPPT.CMV.ffluciferase.WPRE (Penn Vector Core) andcryopreserved in liquid nitrogen. Oneweek before tumor implan-tation, BT474-M1.ffluc cells at passage 56 were thawed, expandedin DMEM/F12 (Corning) with 10% fetal bovine serum and 1%penicillin/streptomycin at 37�C and 5% CO2, and passaged 3days before tumor cell implantation. Normal cell morphologywas confirmed at timeof passage andon theday of harvest.On theday of tumor injection, cells were counted with a Countess IIcytometer (Life Technologies) and suspended at 1� 105 cells per5 mL in 50%/50% (v/v) PBS/MatriGel (Corning).

For the injection procedure, mice were anesthetized with keta-mine/xylazine. Fur on the scalp and neck was sheared. A 1.7-mg,90-day time-release 17-b estradiol pellet (Innovative Research ofAmerica) was implanted subcutaneously in the dorsum of theneck and readministered every 90 days during the study. Micewere fixed in a stereotaxic apparatus. Exposed skin was cleansedwith povidone iodine and 70% ethanol. A 1-cm anterior–poste-rior incision was made over the top of the skull, and the bregmawas identified. A pneumatic drill was used to drill a burr hole inthe skull that was positioned 0.8mmposterior and 2.2mm left ofthe bregma.

A 25-mL syringe (Hamilton Company) was loaded with 5 mL ofcell suspension and positioned in a mechanical injector on thestereotaxic frame. After bringing the needle to 0.8 mm posteriorand 2.2 mm left of the bregma, the needle was moved 4.0 mmdeep into the brain parenchyma and then lifted 1.0mm to create apocket into which to inject cells. The needle was left in place for5 minutes before the cell suspension was mechanically injectedover 10minutes; the needlewas left in place for another 5minutesbefore being removed slowly. After suturing incisions, micerecovered on a 37�C heating pad and were given 100 mL of2 mg/kg enrofloxacin in PBS and 0.3 mg/kg buprenorphine inPBS subcutaneously.

Mice weremonitored daily. Whenmoribund,mice were eutha-nized by overexposure to CO2 followed by cervical dislocation. Atnecropsy, brains and tumor were either snap frozen on dry iceand stored at �80�C for transgene expression and GC analysis,

cryopreserved in optimal cutting temperature medium usingliquid nitrogen, or preserved in formalin.

Tumor volumeTo determine the dose effect of our treatment, we used an

AAV9var vector, which behaves similarly to AAV9 in vivo. Vectorwas administered 2 weeks before tumor implantation. Thirty-fivedays after tumors were implanted, brains were harvested. Bluntdissection at the tumor injection needle track was used to isolatetumors from surrounding brain tissue. Measurement of tumordiameter was performed with digital Vernier calipers (ThermoFisher). The tumor diameter was then measured in 3 dimensions(x, y, and z), and the tumor volume was calculated as the volumeof an ellipsoid, 4/3 � p � x/2 � y/2 � z/2.

ICV Herceptin and AAV9.trastuzumab administration forquantification of trastuzumab in brain tissue

AAV9.trastuzumab was diluted in PBS. A total of 1e11 GC perRag1�/�mouse was delivered ICV in a final volume of 10 mL. Twoweeks later, brains were harvested.

Herceptin was diluted in PBS and administered ICV in a 10 mLvolume at a dose of 15 mg/mouse brain, which is roughly equiv-alent to a 50-mg i.t. dose in humans by brain mass. Brains wereharvested 24, 48, and 168 hours after ICV administration ofHerceptin and homogenized as described above in 1-mL tissuelysis buffer. Protein A enzyme-linked immunosorbent assay(ELISA), performed as described below, was used to determineng/mL of trastuzumab in brain homogenate. Multiplying thistrastuzumab concentration by the homogenate volume of 1 mLyielded the amount of trastuzumab per brain in ng.

The half-life of Herceptin in brain tissue was calculated asfollows:

t1/2 ¼ t ln(2)/(ln (xi) � ln(xt)), where t ¼ time point, xi is theinitial quantity at t ¼ i, and xt is the quantity at time point t.

NK cell depletionPK136 (anti-mouse NK1.1) antibody was purified from PK136

hybridoma supernatant (ATCC HB-191) using the protein Apurification kit (Sigma-Aldrich) and supplemented as neededwith PK136 available commercially (Leinco Technologies). Micewere given 100 mg PK136 or PBS intraperitoneally (i.p.) on days 5and 1 before tumor implantation and then weekly for the dura-tion of the experiment.

Systemic macrophage depletionChlodronate liposomes or PBS liposomes (Nico van Rooijen,

Vrije University Medical Center, Amsterdam, the Netherlands)were injected i.p. at 10 mL/g body weight on days 5 and 1 beforetumor implantation, and then weekly for the duration of theexperiment.

Nonhuman primate studyAn adult female cynomolgus macaque weighing 4.38 kg was

anesthetizedwith ketamine/dexmedetomidine, and the hair over-lying the occiput and dorsal neck was shaved. A spinal needle wasinserted suboccipitally directly into the cisterna magna, and 1mLof CSF was collected. Vector was then infused into the cisternamagna in a volume of 1 mL over 1 minute. The needle wasremoved slowly followed by application of pressure to the punc-ture site. CSF was collected by the same procedure at indicatedtime points over the course of 6 months.

Viral Vector Trastuzumab Therapy Attenuates HER2þ Tumors

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Preparation of serumBlood was collected by retro-orbital or submandibular bleed

into Z-Gel microtube serum separators (Sarstedt) and incu-bated at room temperature for 20 minutes. After centrifugingfor 5 minutes at 5,000 RPM in a tabletop microcentrifuge(accuSpin micro 17, Thermo Fisher Scientific), serum wasstored at �80�C.

Preparation of brain homogenateBrain homogenates were prepared by chipping frozen brain

into tissue lysis buffer (25mmol/L Tris–HCl, 5mmol/L EDTA, 1%Triton-X, 150 mmol/L NaCl, pH 7.6) containing 3 times thenormal concentration of cOmplete Protease Inhibitor CocktailTablets (Roche). Samples were homogenized with stainless-steelbeads on a TissueLyzer II (Qiagen) at 30 Hz for 2 minutes, frozenat �80�C, thawed, and then centrifuged at 17,000 � g for 60minutes at 4�C to remove myelin debris. A bicinchoninic acidassay (ThermoFisher Scientific)wasused todetermine the proteinconcentration of brain homogenates.

Protein A and mac251 enzyme-linked immunosorbent assayProtein A ELISA was used to quantify trastuzumab and 2.10A

mAb expression in serum and brain homogenates. 201IA wasquantified using a specific SIV mac251 gp120 antigen ELISA. Allsteps were performed at room temperature unless otherwisestated. Plates were washed with a 405TS microplate washer(BioTek) with PBS þ 0.05% Tween-20. Protein A (Sigma-Aldrich) was suspended in PBS and stored at �20�C. Costar96-well EasyWash ELISA assay plates (Corning) were coatedwith 5 mg/mL protein A or 2 mg/mL mac251 gp120 in PBSovernight at 4�C and then blocked with PBS þ 0.5% bovineserum albumin (protein A ELISA) or fetal bovine serum (201IAELISA). Samples were diluted in PBS and plated. Herceptin(Roche) or purified 201IA was used as a quantitative standard.For brain homogenate ELISAs, brain homogenate from untreat-ed mice was spiked into the standard curve wells at a dilutionequal to that of samples on the plate. After washing, plates wereincubated with AffiniPure polyclonal goat anti-human IgG-biotin (Jackson ImmunoResearch Laboratories) followed bystreptavidin–horseradish peroxidase (Abcam). Plates weredeveloped with TMB substrate, stopped with 2N H2SO4, andread using a SpectraMax M3 plate reader (Molecular Devices) at450 nm.

Biodistribution by qPCRAAV9 vector GCs in brain were quantified using TaqMan qPCR

(ThermoFisher Scientific). Briefly, frozenbrain tissuewas chippedintoALTBuffer (Qiagen) andhomogenizedwith steel beads usinga TissueLyzerII (Qiagen). Phenol:chloroform:isoamyl alcohol(25:24:1; Sigma-Aldrich) extraction and isopropanol precipita-tion was used to isolate tissue DNA. TaqMan qPCR primers andprobe were designed against the SV40 polyadenylation signal ofthe vector.

HistologyHER2 staining was performed on formalin-fixed, paraffin-

embedded tissue samples. Sections were deparaffinizedthrough a xylene and ethanol series, boiled in a microwavefor 6 minutes in 10 mmol/L citrate buffer (pH 6.0) for antigenretrieval, treated sequentially with 2% H2O2 (15 minutes;Sigma-Aldrich), avidin/biotin blocking reagents (15 minutes

each; Vector Laboratories), and blocking buffer (1% donkeyserum in PBS þ 0.2% Triton for 10 minutes) followed byincubation with rabbit anti-HER2 primary antibody (1 hour;Abcam ab2428) and biotinylated donkey anti-rabbit secondaryantibody (45 minutes; Jackson ImmunoResearch Laboratories)diluted in blocking buffer. Bound antibodies were visualizedwith a Vectastain Elite ABC kit (Vector Laboratories) using DABas the substrate. Sections were counterstained with hematox-ylin to show nuclei.

Immunofluorescence staining for human IgG was performedon cryosections. Sections were fixed in 4% paraformaldehyde inPBS for 10minutes, permeabilized and blocked in 0.2% Triton inPBS containing 1% donkey serum for 30 minutes, and incubatedfor 1 hour with a goat antibody against the crystallizable fragment(Fc) of human IgG (Jackson ImmunoResearch Laboratories#109-005-098) diluted in 1%donkey serum inPBS. Afterwashingsections in PBS, bound primary antibodies were detected withfluorescein isothiocyanate-labeled secondary donkey anti-goatantibodies (Jackson ImmunoResearch Laboratories) diluted in1% donkey serum in PBS. After washing in PBS, sections weremountedwith Vectashield (Vector Laboratories) containingDAPIas a nuclear counterstain.

IHC to detect NK cells and HER2 was also performed oncryosections. Sections for NK cell staining were fixed in acetoneat �20�C for 7 minutes, air dried, sequentially treated with0.3% H2O2 in PBS for 10 minutes, avidin/biotin blockingreagents (15 minutes each; Vector Laboratories), and blockingbuffer (1% donkey serum in PBS, 20 minutes). Sections forHER2 staining were fixed in 4% paraformaldehyde in PBS for10 minutes, permeabilized in 0.2% Triton in PBS for 30minutes, and sequentially treated with 0.3% H2O2 in PBS(10 minutes) and avidin/biotin blocking reagents (15 minuteseach; Vector Laboratories). Sections were then blocked with 1%donkey serum in PBS for 20 minutes, treated with primaryantibody (1 hour), and corresponding biotinylated secondaryantibody (45 minutes; Jackson ImmunoResearch Laboratories)diluted in 1% donkey serum in PBS. Primary antibodies were asfollows: rat anti-mouse Ly-49G2 (clone 4D11, BD Pharmingen,BD Biosciences) for NK cells or rabbit anti-mouse Erb2 (Abcamab2428) for HER2. A Vectastain Elite ABC kit (Vector Labora-tories) was used according to the manufacturer's instructionswith DAB as the substrate.

To detect NK cells on formalin-fixed, paraffin-embeddedsections, in situ hybridization (ISH) was performed using theViewRNA ISH Tissue Assay Kit (Thermo Fisher Scientific)according to the manufacturer's protocol. Z-shaped probe pairsspecific for mouse Ncr1 (natural cytotoxicity triggering receptor1, NKp46) RNA were synthesized by the kit manufacturer. Thedeposition of Fast Red precipitates indicating positive signalswas imaged by fluorescence microscopy using a rhodaminefilter set. Sections were counterstained with DAPI to shownuclei.

ResultsIT AAV9.trastuzumab can prevent or treat tumors in a novelRag1�/� orthotopic xenograft model of HER2þ BCBM

I.t.-administered trastuzumab can increase survival and delaytumor progression in patients with HER2þ BCBM. However,trastuzumab has been reported to have a half-life of 12 hours inhuman CSF (21, 25). We aimed to administer AAV9.trastuzumab

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i.t., enabling constitutive secretion of trastuzumab by neuronsand astrocytes in situ, better tumor exposure to the immunother-apy, and thus longer median survival.

We began by developing a novel xenograft mouse model tostudy the effect of i.t. AAV9.trastuzumab against HER2þ BCBM.In order to ensure our model had no endogenous IgG and was

not "leaky" with respect to immune cell development, we choseto use Rag1�/� mice. As trastuzumab does not bind to murineHER2, using a xenografted cell line that expresses human HER2was necessary for these experiments. Therefore, we selected theHER2þ human ductal carcinoma cell line BT474.M1.ffluc foruse in our experiments. Importantly, BT474.M1.ffluc cells are

Figure 1.

Intrathecal AAV9.trastuzumabtumor prophylaxis. A, Kaplan–Meier survival curves for mice thatreceived i.t. AAV9.trastuzumabtumor prophylaxis, i.t.AAV9.2.10AmAb-negative controltreatment, or no treatment at least21 days before tumor challenge. B,Tabulated survival and statisticaldata for A. C and D, Serumexpression of trastuzumab and2.10A mAb measured by protein AELISA. E, IgG transgeneexpression in brain tissuemeasured by protein A ELISA ofbrain homogenate and graphed asa percentage of total protein inbrain homogenate.






relatively sensitive to trastuzumab treatment, making detectionof small changes in efficacy after administration of immuno-modulatory elements possible. We used these cells across all ofour experiments in order to allow continuity between efficacystudies and studies conducted to determine the mechanism ofour treatment.

To establish the model, we administered BT474.M1.ffluc cells(100,000) stereotactically into the caudate/putamen region of themouse brain. We originally monitored tumor bioluminescencetwice weekly following i.p. administration of luciferin. However,we found that the administration of luciferin concurrent with thepresence of the subcutaneous 17-beta estradiol time-release pel-lets, used routinely to enhance engraftment, led to significanturinary and kidney pathology, requiring euthanasia of animals.We therefore ceased luciferase imaging for the remainder of ourexperiments and instead used survival and tumor volume as ourprimary endpoints.

We first tested AAV9.trastuzumab as HER2þ CNS tumor pro-phylaxis in our Rag1�/� xenograft model. In a pilot experiment,we administered AAV9.trastuzumab, AAV9.201IA (a negativecontrol vector expressing a rhesus antisimian immunodeficiencyvirus IgG immunoadhesin), AAV9.null (AAV9 vector with anintact genome and expression cassette but no transgene), or PBSi.t. by ICV injection into Rag1�/� mice at least 3 weeks beforestereotactic BT474.M1.ffluc tumor implantation (100,000 cells).Mice that receivedAAV9.trastuzumab tumor prophylaxis survivedsignificantly longer than mice that received control treatments[Supplementary Fig. S1A and S1B; 111 days vs. 48.5 days (PBScontrol); P ¼ 0.0012]. Importantly, the median survival amonggroups of mice that received control treatments did not signifi-cantly differ.

We next repeated this experiment with additional mice in eachtreatment group and using a full-length IgG-negative controlvector (AAV9.2.10AmAb). We administered AAV9.trastuzumabor AAV9.2.10AmAb i.t. by ICV injection at least 21 days beforetumor implantation. Untreated mice served as an additionalcontrol. We implanted HER2þ BT474-M1.ffluc cells into thebrain parenchyma stereotaxically and monitored mice dailyuntil moribund. Kaplan–Meier survival curves indicated that themedian survival of mice that received i.t. AAV9.trastuzumabtumor prophylaxis was significantly greater than mice thatreceived AAV9.2.10AmAb (Fig. 1A and B; 124 days vs. 46.5 days;P < 0.0001, hazard ratio ¼ 0.0306). The median survival of micethat received AAV9.2.10AmAb was not significantly differentfrom mice without treatment (Fig. 1A and B; 46.5 days vs. 50days; P ¼ 0.4306, hazard ratio ¼ 1.1868). Protein A ELISAquantification of IgG transgene expression in serum (Fig. 1C andD) and brain tissue homogenate, normalized to total protein inbrain homogenate (Fig. 1E), indicated that transgene expressionwas similar between groups that received AAV9.trastuzumab andAAV9.2.10AmAb. Biodistribution analysis of AAV9 vector GCs inbrain tissue demonstrated equivalent genome depositionbetween groups that received vector (Supplementary Fig. S2A).Tumors remainedHER2þ at the time of necropsy (SupplementaryFig. S2B).

To determine if i.t. AAV9.trastuzumab can serve as atreatment for existing HER2þ BCBM, we implanted HER2þ

BT474-M1.ffluc tumors into Rag1�/� mice and administeredi.t. AAV9.trastuzumab or no treatment 3 days after tumorimplantation. Kaplan–Meier survival curves indicated that themedian survival of mice that received AAV9.trastuzumab was

significantly greater than mice that received no treatment (Sup-plementary Fig. S3A and S3B; 82 days vs. 61 days; P ¼ 0.002).

Dose escalation of AAV9var.trastuzumab leads to smaller day35 tumor volume

We next performed a dose-escalation tumor prophylaxis studyusing a closely related variant of AAV9 that performs similarlyin vivo. We administered 4 doses of AAV9var.trastuzumab 2weeksbefore tumor implantation. A control group received no treat-ment. At day 35 after tumor implantation, we harvested brainsand carefully dissected tumors from surrounding tissue. Wemeasured the tumors in 3 dimensionswith digital Vernier calipersand calculated the tumor volume. The mean volume of tumorsdecreased as the dose of AAV9var.trastuzumab increased, andmice that received no treatment had the largest mean tumorvolume (Fig. 2A and B).

Secreted trastuzumab binds to HER2þ brain tumorsWe next used anti-IgG immunofluorescence of day 47 tumor

cryosections to determine if secreted trastuzumab could bind toHER2þ tumor cells in vivo. Micrographs showed trastuzumabdecorating tumors frommice that received i.t. AAV9.trastuzumabbut not tumors frommice that received i.t. AAV9.2.10AmAbor notreatment (Fig. 3; component channels and merge are shown inSupplementary Fig. S4). Additionally, neurons transduced by

Figure 2.

Day 35 tumor volume following AAV9var.trastuzumab tumor prophylaxis.A, Tumor volumes from mice that received the indicated doses of AAV9var.trastuzumab tumor prophylaxis or no treatment. Tumor volume wasmeasured 35 days after tumor implantation. B, Tabulated results andstatistical data for A.

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AAV9.trastuzumab or AAV9.2.10AmAb stained positive for IgG.Secreted trastuzumab also decorated day 20 tumors from micethat received AAV9.trastuzumab (Supplementary Fig. S5).

NK cells mediate i.t. AAV9.trastuzumab tumor prophylaxisTrastuzumab exerts its effect against peripheral tumors mainly

by facilitating antibody-dependent cell-mediated cytotoxicity(ADCC) of HER2þ tumor cells (28–30). We hypothesized thatthe same mechanism would govern AAV9.trastuzumab tumorprophylaxis in the CNS. We first evaluated AAV9.trastuzumabtumor prophylaxis in NSG mice, which lack NK cells and func-tional macrophages, both of which can perform antibody-depen-dent tumor cytotoxicity. As expected, AAV9.trastuzumab tumorprophylaxis failed in NSG mice (Supplementary Fig. S6A–S6C).Survival was comparable between groups that received no treat-ment (37 days) or AAV9.trastuzumab (40 days, P ¼ 0.862).Transgene expression in brain tissue was similar among micethat received vector.

We next sought to determine if NK cells or macrophagesplayed a larger role in i.t. AAV9.trastuzumab tumor prophylaxisin the CNS. To do so, we first used continuous NK cell depletionby i.p. administration of the anti-NK1.1 antibody PK136 in ourRag1�/� xenograft tumor model (Fig. 4A and B). Mice thatreceived AAV9.2.10AmAb with or without NK cell depletionsurvived a median of 53 and 50 days, respectively. Mice that

received AAV9.trastuzumab tumor prophylaxis and no NK celldepletion lived a median of 156 days. The median survival ofmice that received AAV9.trastuzumab tumor prophylaxis withcontinuous NK cell depletion was significantly shorter (73 days;P < 0.0001, compared with AAV9.trastuzumab without NK celldepletion).

We subjected the formalin-fixed tumors from mice in the NKcell depletion experiment to ISH for NK cell–specific Ncr1(NKp46) RNA. Micrographs indicate that NK cells infiltrated onlytumors from mice given AAV9.trastuzumab without NK celldepletion (Fig. 4C; Supplementary Fig. S7). IgG transgene expres-sion in brain tissue homogenate was comparable between groupsthat received vector (Supplementary Fig. S8A). Ncr1 ISH ofspleens confirmed successful depletion of NK cells (Supplemen-tary Fig. S8B). We also cryosectioned day 20 tumors from micethat received prophylactic AAV9.trastuzumab, AAV9.2.10AmAb,or no treatment; this experiment was conducted without NK celldepletion. IHC staining for Ly-49G2 confirmed that NK cellsinfiltrated day 20 tumors only from mice that received AAV9.trastuzumab tumor prophylaxis (Fig. 5).

To determine if systemically circulating macrophages play arole in AAV9.trastuzumab tumor prophylaxis in our model, weused continuous macrophage depletion by i.p. administration ofclodronate liposomes. Mice that received no treatment with orwithout macrophage depletion survived a median of 53 and 50

Figure 3.

IgG immunofluorescence of day 47 tumor cryosections. Green channel (human/rhesus IgG) overexposures of day 47 tumors from mice that prophylacticallyreceived i.t. AAV9.trastuzumab, i.t. AAV9.2.10AmAb, or no treatment. Staining of tumors from mice that received AAV9.trastuzumab and AAV9.2.10AmAbshows neurons expressing IgG (arrows) and tumors decorated with trastuzumab. Tumors from mice that received AAV9.2.10AmAb were not decoratedby IgG. DAPI (blue), tumor (T), and brain parenchyma (B).






days, respectively (Supplementary Fig. S9A and S9B). Mice thatreceived AAV9.trastuzumab without macrophage depletion sur-vived a median of 92 days (P � 0.0001 compared with mice thatreceived no treatment without macrophage depletion). Mice thatreceived AAV9.trastuzumab with macrophage depletion surviveda median of 70.5 days (P ¼ 0.3268, compared with AAV9.trastuzumab without macrophage depletion). IgG transgeneexpression in brain tissue was comparable among groups that

received vector (Supplementary Fig. S9C), andmacrophage deple-tion was confirmed by IHC staining of spleens for CD68 (Sup-plementary Fig. S9D).

Comparisonof trastuzumab levels inbrain after administrationof i.t. Herceptin or i.t. AAV9.trastuzumab

To compare the amount of trastuzumab in brain tissue afterICV administration of AAV9.trastuzumab versus Herceptin, we

Figure 4.

Intrathecal AAV9.trastuzumab tumorprophylaxis in the setting of continuousNK cell depletion. A, Kaplan–Meiersurvival curves for mice that receivedi.t. AAV9.trastuzumab tumorprophylaxis or i.t. AAV9.2.10AmAbcontrol treatment with or withoutcontinuous NK cell depletion usingPK136 (anti-NK1.1 antibody). B,Tabulated survival and statistical datafor Fig. 5A. C, In situ hybridization forNcr1 (NKp46) RNA (red) in formalin-fixed paraffin-embedded tumorsharvested at necropsy from theexperiment in Fig. 5A. Larger images ofthe AAV9.2.10AmAb-treated tumorsare shown in Supplementary Fig. S7.DAPI (blue), tumor (T), and brainparenchyma (B).

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administered 1e11 GC AAV9.trastuzumab or 15 mg HerceptinICV to Rag1�/� mice. We harvested brains from mice thatreceived AAV9.trastuzumab 2 weeks after vector administra-tion, and brains that received Herceptin 24, 48, and 168 hoursafter administration. Figure 6A indicates that the medianamount of trastuzumab expressed in brain tissue in mice thatreceived AAV9.trastuzumab is equivalent to the amount ofHerceptin in brain tissue 24 hours after administration of 15mg antibody. Over time, the amount of Herceptin in braintissue decreased, as expected. We calculated the half-life ofHerceptin in mouse brain tissue to be 42 hours between the24- and 48-hour intervals and 46 hours between the 48- and168-hour intervals.

Vector-mediated antibody expression in macaque CSFTo examine the safety of using an AAV9 vector to express an

antibody in the CNS of a nonhuman primate, we delivered 1 �1012 GC/kg of AAV9.201IA i.t. into the cisternamagna of a femalecynomolgus macaque. We collected CSF at regular intervals overthe next 6 months and quantified 201IA expression in CSF byELISA. Results in Fig. 6B show a peak concentration of 201IA inCSFof 0.350 mg/mL at day 48,which plateaued to 0.165mg/mLbyday 77 and 0.153 mg/mL by the end of the study (day 168).Importantly, no serious adverse events occurred after adminis-tration of vector.

DiscussionMetastasis of HER2þ breast cancer to the brain is a devastating

diagnosis with a poor prognosis due to a lack of targeted andeffective treatments. Although i.t. administration of trastuzumabhas been reported to slow disease progression and prolongsurvival, the benefit is modest, likely due to the rapid turnoverof CSF reported by others, resulting in poor tumor exposure toantibody. An unmet need for effective treatments exists forpatients with this disease.

The advent of AAV vectors for gene transfer has revolution-ized the field of gene therapy. The discovery of CNS-tropicvectors, such as AAV9, represents a boon for clinicians seekingto localize biological treatments behind the blood–brain bar-rier. A single i.t. administration of AAV9 vector into the CSF byway of the lateral ventricles or cisterna magna leads to wide-spread transduction of neurons and astrocytes throughout thecortex, cerebellum, and spinal cord, resulting in long-livedproduction of transgene product in the CNS compartment(26, 27, 31).

We successfully demonstrated that AAV9.trastuzumabadministered i.t. at a moderate vector dose, both as tumorprophylaxis and as tumor treatment, significantly extendsmedian survival in a xenograft mouse model in which thehuman HER2þ cancer cell line BT747-M1.ffluc is implantedinto the brains of Rag1�/� mice. In the prophylactic setting, the

Figure 5.

NK cell IHC staining of day 20 tumors. Mice received AAV9.trastuzumab, AAV9.2.10AmAb, or no treatment 21 days before tumor implantation. Tumors wereharvested 21 days after implantation, and IHC staining for NK cells (Ly-49G2) was performed on sample cryosections. Tumor (T), brain parenchyma (B), and necrotictumor (N).






median survival was 2.48 times greater for mice that receivedAAV9.trastuzumab tumor prophylaxis than for mice thatreceived control AAV vector treatment or no treatment. Addi-tionally, dose-escalation studies using AAV9var.trastuzumabtumor prophylaxis indicated that higher doses of vector led tosmaller tumors 35 days after implantation. In the treatmentsetting, the median survival benefit from AAV9.trastuzumabadministration was significant but less, perhaps in part due tothe shorter total duration of exposure of tumor cells toexpressed trastuzumab.

Our studies also indicate that trastuzumab secreted by neu-rons and astrocytes maintains its antigen specificity and effectorfunction despite the fact that these cells do not normallyproduce IgG. Using immunofluorescence microscopy, weshowed that HER2þ tumors from mice treated with AAV9.trastuzumab, but not AAV9.2.10AmAb, are decorated withexpressed IgG.

The effector functions of trastuzumabhave beenwidely studied(28, 29). Trastuzumabbound toHER2on tumor cells (i)mediatesADCCwhen its Fc binds to activating Fcg receptors (e.g., FcgRIIIa)on immune cells such as NK cells and macrophages; (ii) preventsinitiation of signal transduction when HER2 homodimerizes orheterodimerizes with other members of the HER family, thusslowing tumor growth; (iii) blocks proteolytic cleavage of theHER2 ectodomain, preventing the remaining membrane-boundp96 protein from activating progrowth signaling pathways; (iv)disrupts proangiogenic pathways; and (v) disrupts DNA repairpathways.

The predominant mechanism by which trastuzumab actsagainst peripheral HER2þ tumors in patients is ADCCby immunecells carrying Fcg receptors, such asNK cells andmacrophages.Wehypothesized that the same would be the case in our xenograftmodel. AAV9.trastuzumab tumor prophylaxis failed whenadministered to NSG mice, which lack NK cells and functionalmacrophages, compared with Rag1�/� mice; this result suggestsan NK cell- or macrophage-mediated mechanism of tumor pro-phylaxis. When we depleted NK cells in the Rag1�/� model, themedian survival of mice decreased substantially, indicating thatNK cell–driven ADCC of tumor cells is responsible for a majorityof the survival benefit. Additionally, immunofluorescence andIHC staining of NK cells in tumors indicated that NK cellsinfiltrated only into tumors of mice that received AAV9.trastuzu-mab tumor prophylaxis, not in tumors from untreated or control-treated mice. These NK cells were present in tumors as early as 20days after tumor implantation and as late as the time of necropsy.To our knowledge, this is the first report revealing the predom-inant mechanism by which trastuzumab works against HER2þ

tumors in the CNS.Ourmacrophage depletion studies indicated that these cells do

not contribute significantly to the antitumor effect of AAV9.trastuzumab tumor prophylaxis in this model. However, as clo-dronate liposomes do not cross the blood–brain barrier, theseexperiments can only address whether systemically circulatingmacrophages play a role in AAV9.trastuzumab tumor prophylax-is. Tissue-resident macrophages/microglia may still contribute asmall part to the antitumor effect of AAV9.trastuzumab in thismodel.

Wewereunable to administer i.t.Herceptin as apositive controlin our experiments due to Institutional Animal Care and UseCommittee restrictions, which prohibit us from performingmorethan a single ICV injection during the lifetime of a mouse. In lieuof this, we report that the half-life of Herceptin in the brain tissueof Rag1�/� mice is around 42 to 46 hours. We also showed thatthemedian amount of trastuzumab in brains ofmice 2weeks afterAAV9 vector administration was equivalent to the amount inbrains 24 hours after receiving 15 mg by ICV injection. Finally, wewere able to demonstrate safe, constant expression of an antibodyin the CNS of a nonhuman primate for 6 months after ICMadministration of AAV9 vector.

The BT474.M1 cell line highly overexpresses HER2, meaningour results likely overestimate the response to our therapy inhumans given the heterogeneity of HER2 expression both withintumors and among different HER2þ BCBM in patients. Althoughusing other cell lines in thismodelmay shed light on the expectedefficacy of our treatment in humans, our results illustrate proof ofconcept that AAV9.trastuzumab can reduce tumor burden andincrease survival when administered to mice with a HER2þ breastcancer brain tumor. It is also important to note that patients

Figure 6.

Trastuzumab quantitation in the CNS of mice and a cynomolgus macaque. A,Trastuzumab quantified in brain tissue at the time points indicated followingeither a 15 mg ICV bolus of Herceptin or 2 weeks after ICV AAV9.trastuzumabvector administration (1e11 GC/mouse). B, Expression of 201IA in the CSF of acynomolgus macaque that received AAV9.201IA by intracisterna vectoradministration (1e12 GC/kg).

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whose tumors become resistant to trastuzumab treatment wouldlikely not benefit from our therapy. This will be an importantconsideration when choosing a target population for clinicalstudies and treatment.

Additionally, ourmice bore onebrain tumor, but patients oftendevelop multiple metastases. Testing AAV9.trastuzumab tumorprophylaxis in a model where tumor cells are administered viacarotid artery injection would indicate whether our treatment iseffective against multiple brain tumors. We also used AAV9.trastuzumab in these studies as a monotherapy. Unsurprisingly,all mice eventually succumbed to outgrowth of tumors, whichremain HER2þ at necropsy. Combining AAV.trastuzumab withchemotherapy and/or radiotherapy will likely be synergisticagainst HER2þ BCBM, both in this xenograft model and inpatients.

Trastuzumab infusion is standard of care for patients withHER2-overexpressing breast cancer, and i.t. delivery of trastuzu-mab has been shown to be beneficial. However, trastuzumabexpression cannot be turned off after AAV9-mediated gene trans-fer, although there are efforts in the gene therapy field to developan inducible expression system. Related to this, cardiotoxicity is aknown side effect that occurs in a small subset of patients whoreceive systemic trastuzumab treatment. Risk factors for cardio-toxicity have been identified, including previous anthracycline-containing chemotherapeutic regimens, age, and existing cardiacdysfunction (32–34).

After i.t. administration of i.t. AAV9.trastuzumab, we anticipatea lower concentration of trastuzumab in blood than after i.v.administration of Herceptin. This would be less likely to lead tocardiotoxicity. Regardless, women with CNS metastasis fromHER2þ breast cancer almost always have systemic disease, neces-sitating treatment with systemic administration of trastuzumabwhere safety monitoring of cardiac function is standard. We willincorporate this aspect into the designof ourfirst-in-humanphaseI safety trial.

Given this fact and the results of our experiments, we intend tomove AAV9.trastuzumab toward the clinic after successful com-pletion of necessary preclinical studies, including safety andtoxicology experiments in large animal models. We plan to firstassess the safety, efficacy, and pharmacokinetics of AAV9.trastu-zumab in women with documented CNS lesions from HER2þ

breast cancer. Subject to safety and efficacy in this setting, we willsubsequently assess AAV9.trastuzumab in a similar patient pop-ulation with systemically metastatic HER2þ disease prior toclinical or radiologic diagnosis of CNS lesions. Given that AAV

transgene expression has been documented to persist for years innonhuman primates and humans (35, 36), this approach has thepotential to be an integral part of the adjuvant therapy for patientswith early diagnosis of HER2þ breast cancer after curative resec-tion of the breast lesion. Indeed, our AAV platform has thepotential to be expanded to express other therapeutic immu-notherapies behind the blood–brain barrier to treat CNS diseasesand address other unmet therapeutic needs.

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

Authors' ContributionsConceptionanddesign:W.T.Rothwell, P. Bell,M.P. Limberis,M. Li, J.M.WilsonDevelopment of methodology: W.T. Rothwell, P. Bell, L.K. Richman,M.P. Limberis, M. LiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. BellAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W.T. Rothwell, P. Bell, J.M. WilsonWriting, review, and/or revision of the manuscript: W.T. Rothwell, P. Bell,M.P. Limberis, J.M. WilsonAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L.K. Richman, A.P. Tretiakova, M. LiStudy supervision: W.T. Rothwell, J.M. WilsonOther (oversaw histology work): P. Bell

AcknowledgmentsWe would like to thank Lewis Chodosh and Jason Ruth for their generous

gift of BT474.M1 cells; Lewis Chodosh, Jos�e Conejo-Garcia, Chi Van Dang,and Laura A. Johnson for their guidance and advice; Tamara Goodefor procedural instruction and advice with the xenograft model; DeirdreMcMenamin, Christine Draper, and the Penn Gene Therapy Program (GTP)Program for Comparative Medicine for procedural assistance; JamunabaiPrakash and the Penn GTP Cell Morphology Core for assistance withhistopathology; the Penn Vector Core; Christian Hinderer for advice anddirection; Jenny Greig for her generous gift of AAV9.2.10AmAb and foradvice and direction; and Tarek Sahmoud for his assistance with the clinicaldevelopment plan.

This work was funded by the University of Pennsylvania Gene TherapyProgram.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 2, 2018; revised July 9, 2018; accepted August 23, 2018;published first August 28, 2018.

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2018;78:6171-6182. Published OnlineFirst August 28, 2018.Cancer Res William T. Rothwell, Peter Bell, Laura K. Richman, et al. Inhibits Tumor Growth of Human HER2-Positive Xenografts in MiceIntrathecal Viral Vector Delivery of Trastuzumab Prevents or

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Intrathecal Viral Vector Delivery of ... - Cancer ResearchWilliam T. Rothwell1, Peter Bell1, Laura K. Richman1, Maria P. Limberis1, Anna P.Tretiakova1, Mingyao Li1,2, and James M.Wilson1