[CANCER RESEARCH 59, 6159–6163, December 15, 1999]

Imaging Brain Tumors by Targeting Peptide Radiopharmaceuticals through theBlood-Brain Barrier 1

Atsushi Kurihara and William M. Pardridge 2

Department of Medicine, University of California at Los Angeles School of Medicine, Los Angeles, California 90095

ABSTRACT

Present day imaging of brain tumors requires a disrupted blood-brain barrier (BBB). However, the BBB is intact in the early stages ofbrain tumor growth, when diagnosis is most critical. Relative to normalbrain, brain tumor cells frequently overexpress peptide receptors, suchas the receptor for epidermal growth factor (EGF). Peptide radiophar-maceuticals such as radiolabeled EGF could be used to image earlybrain tumors, should these radiopharmaceuticals be made transport-able through the BBB. The present studies describe a bifunctionalmolecule that contains both biologically active human EGF radiola-beled with 111In and an anti-transferrin receptor monoclonal antibodythat undergoes transcytosis through the BBB via the endogenous trans-ferrin transport system. The two domains of the bifunctional conjugateare separated by aM r 3400 polyethyleneglycol linker, which releasessteric hindrance and allows the conjugate to bind to both the EGFreceptor, to image the brain tumor, and to the transferrin receptor, toenable transport through the BBB. Successful imaging of experimentalbrain tumors with this system is demonstrated in nude rats bearingcerebral implants of human U87 glioma.

INTRODUCTION

Human brain tumors are the leading cause of cancer deaths ofpersons 15–34 years of age (1). Fatality rates could be diminishedwith early diagnosis of human brain tumors. However, present-dayimaging modalities of brain tumors require a disrupted BBB,3 and theBBB is generally intact in human brain tumors in the early stages (2,3). Moreover, the brain adjacent tumor, which is the site of tumorgrowth and extension into normal brain, has an intact BBB (4). Thismakes it difficult to detect residual glioma in the perioperative orpostoperative state with present-day imaging modalities. The biolog-ical properties of human gliomas include overexpression of peptidereceptors relative to normal brain. Human gliomas overexpress afunctional receptor for EGF (5, 6). On this basis, radiolabeled EGFhas been proposed as a peptide radiopharmaceutical for imaging braintumors (7). However, EGF does not cross the BBB (8) and cannotimage brain tumors in the early stage when treatment is most needed,and the BBB is intact. Similarly, MAbs to the EGFR have beenproposed as antibody radiopharmaceuticals for imaging human glio-mas (9), but these also do not cross the BBB (10). In brain tumorsectionsin vitro, EGF labels human gliomas to a greater extent thanthat achieved with the EGF receptor MAbs (11).

EGF can be transported across the BBB in brain tumors if thispeptide radiopharmaceutical is conjugated to a BBB drug deliverysystem (12). The latter is comprised of a peptide or a peptidomimetic

MAb that undergoes receptor-mediated transcytosis through the BBBvia one of several endogenous peptide transport systems localizedwithin the brain capillary endothelial plasma membrane, which formsthe BBB in vivo. The OX26 murine MAb to the TfR undergoesreceptor-mediated transport through the BBB via the endogenoustransferrin transport system (13, 14). Peptides that are not normallytransported through the BBB, such as EGF, may be conjugated tobrain drug delivery vectors, such as the OX26 MAb, using avidin-biotin technology (12). In this approach, the nontransportable peptideis monobiotinylated in parallel with the preparation of a conjugate ofthe OX26 MAb and SA, and this conjugate is designated OX26/SA.Previous studies (15) showed that EGF could be monobiotinylatedwith retention of high affinity for the human EGF receptor, when thebiotin was attached to the EGF via a 14-atom bis (aminohexanoyl)linker, designated-XX-. However, when the EGF-XX-biotin wasbound to OX26/SA, there was no significant binding to the EGFreceptor because of steric hindrance caused by binding of the EGF tothe brain delivery vector (15). The steric hindrance could be removedby replacement of the 14-atom-XX-linker with a.200-atom linkercomprised of PEG ofMr 3400 molecular weight, designated PEG3400.When the EGF-PEG3400-biotin was bound to OX26/SA, there wasretention of high-affinity binding of the conjugate to the EGFR. ThePEG3400 linker restored the bifunctionality of the conjugate, and theconjugate bound both the EGF receptor, for imaging brain tumors, andthe BBB transferrin receptor, for mediating BBB transport (15).Therefore, the present studies were designed to test the hypothesis thatexperimental brain tumors expressing the human EGFR could beimaged in vivo with an EGF peptide radiopharmaceutical that isenabled to undergo transport through the BBB because of conjugationto a peptidomimetic MAb that transcytoses through the BBB. HumanU87 glioma cells were used to form experimental brain tumors innude rats. The EGF was conjugated with DTPA to enable radiolabel-ing with the111In radionuclide. The EGF that is dual conjugated withDTPA and PEG3400-biotin is designated [111In]-labeled DTPA-EGF-PEG3400-biotin.

MATERIALS AND METHODS

Synthesis of [111In]-labeled DTPA-EGF-PEG3400-biotin and Conjuga-tion to OX26/SA MAb. The technique used to prepare [111In]-labeled DTPA-EGF-PEG3400-biotin conjugated with OX26/SA has been described (12, 15).NHS-PEG3400-biotin was reacted with EGF at room temperature for 60 min ina molar ratio of 5:1, where NHS isN-hydroxysuccinimide and PEG3400 ispolyethyleneglycol ofMr 3400. This mixture was reacted with DTPA dianhy-dride for 60 min in a molar ratio of 50:1. EGF, containing a single DTPA groupand a single PEG3400-biotin, and designated DTPA-EGF-PEG3400-biotin, waspurified by two Superose 12HR fast protein liquid chromatography columns inseries, followed by111In chelation to the DTPA group. The conjugate of theOX26 MAb and recombinant SA, designated OX26/SA, was prepared with athiol-ether linkage (16). Thiolated OX26 was reacted withm-maleimidoben-zoyl N-hydroxysuccinimide ester-activated SA. The number of biotin bindingsites per OX26/SA conjugate was 3.36 0.3, as determined with a [3H]biotinbinding assay (16). The conjugate of [111In]-labeled DTPA-EGF-PEG3400-biotin and OX26/SA was prepared by mixing in a molar ratio of 1:1.6.

U87 MG Radioreceptor Assay.The human glioblastoma multiforme cellline U87 MG was obtained from the American Type Culture Collection(Rockford, MD) and was grown in monolayer culture (37°C, 5% CO2) in

Received 5/10/99; accepted 10/19/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Department of Energy. A. K. was supported bySankyo Co., Ltd., Tokyo, Japan.

2 To whom requests for reprints should be addressed, at Department of Medicine,UCLA School of Medicine, Los Angeles, CA 90095-1682. Phone: (310) 825-8858; Fax:(310) 206-5163; E-mail: wpardridge@med.ucla.edu.

3 The abbreviations used are: BBB, blood-brain barrier; EGF, epidermal growthfactor; EGFR, EGF receptor; MAb, monoclonal antibody; TfR, rat transferrin recep-tor; SA, streptavidin; PEG, polyethylene glycol; DTPA, diethylenetriaminepentaaceticacid; QAR, quantitative autoradiography.

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MEM with 1 mM sodium pyruvate and 10% fetal bovine serum in 24-wellcluster dishes. Cells were washed with 0.01M HEPES, 0.15M NaCl (pH 7.4),and 0.1% BSA (HBSB buffer), followed by 15–120 min incubation at 37°Cwith 200ml of HBSB buffer containing 1.0mCi/ml (0.23 nM) of [111In]-labeledDTPA-EGF-PEG3400-biotin with and without BBB transport vector OX26/SAin the presence and absence of 1mM unlabeled EGF. After incubation,supernatants were aspirated, and the cells were washed two times with cold0.01M HEPES, 0.15M NaCl (pH 7.4) and solubilized by the addition of 0.5 mlof 1 N NaOH and incubation at 37°C for 4 h.111In radioactivity was countedby gamma counter, and protein content of the cells was measured with thebicinchoninic acid protein assay (Pierce Chemical Co., Rockford, IL). Cell-associated binding (surface-binding and intracellular accumulation) was ex-pressed as a percentage of medium radioactivity bound per mg of cell protein(12).

Intracerebral Tumor Implantation and in Vivo Autoradiography. Maleathymic nude rats (Hsd:RH-rnu) weighing 180–210 g were purchased fromHarlan Sprague Dawley (Indianapolis, IN), and were implanted with U87 MGcells. A total of 15 animals was used for this study; 12 animals were examinedwith QAR, and 10 animals were investigated with immunocytochemistry. Aburr hole was drilled 3 mm to the right of midline and 1 mm posterior tobregma. U87 MG cells were suspended in serum-free MEM containing 1.2%methylcellulose. Fiveml of cell suspension (33 105 cells) were injected intothe right caudate nucleus at a depth of 4 mm over 10 s, using a 10-ml Hamiltonsyringe with fixed needle. The syringe was withdrawn after 5 min. At 16 daysafter U87 MG tumor cell implantation, nude rats were i.v. injected with either[111In]-labeled DTPA-EGF-PEG3400-biotin (100mCi) with OX26/SA and 16nmol of unlabeled EGF or [111In]-labeled DTPA-EGF-PEG3400-biotin (100mCi) alone. The coinjection of unlabeled human EGF saturates peripheraluptake of the peptide radiopharmaceutical via the hepatic EGFR, making moreradiopharmaceutical available for uptake by the brain (12). Two h after isotopeinjection, nude rats were sacrificed, and the brain was rapidly removed fromthe cranium and sectioned into 3-mm slabs. The slabs were plunged intopowdered dry ice for rapid freezing over 30 min. Frozen sections of 15mmwere cut with a Bright cryostat and thaw-mounted onto glass coverslips. Afterdrying, these coverslips were placed in apposition to Kodak Biomax MS X-rayfilm and exposed for 6 days at270°C with an intensifying screen. The filmwas scanned with a Hewlett-Packard ScanJet IIcx/T flatbed scanner andtransferred to Adobe Photoshop on a Power Macintosh 7100/66 microcom-puter, followed by colorization with NIH image software, and prints weregenerated with a Kodak printer. The grayscale image of the brain was quan-tified with NIH Image, and means6 SE was computed from three separatemeasurements.

Immunocytochemistry. The expression of the EGF receptor in the U87MG glioblastoma multiforme was investigated with immunocytochemistryboth in cell culture andin vivo as brain tumors in athymic nude rats. The U87MG cells cultured in a 35-mm dish were fixed in 100% methanol (220°C for20 min). The 15-mm frozen sections of experimental tumor in athymic nude ratbrain were fixed in 100% acetone (220°C for 20 min). Endogenous peroxidaseactivity was inactivated with 0.5% H2O2 for 5 min at room temperature, andthe cells were blocked with 3% horse serum for 30 min at room temperature.Two different primary antibodies were used with comparable results. Theantihuman EGFR mouse monoclonal antibody (Upstate Biotechnology, LakePlacid, NY) was used at 10mg/ml. A second primary antibody used in thisstudy was the 528 mouse monoclonal antibody to the human EGFR that hadbeen generated from conditioned medium produced from 528 hybridoma cells,which were obtained from the American Type Culture Collection. The 528antibody has been demonstrated in previous studies to illuminate the humanEGFR of U87 MG cells in nude mice with immunocytochemistry (17). Asisotype controls, mouse IgG1 and mouse IgG2a were used in parallel. Afterreaction with a biotinylated horse antimouse antibody that had been preab-sorbed with rat immunoglobulin, the specimens were exposed to avidin andbiotinylated peroxidase using the Vector ABC method (Vector Labs,Burlingame, CA).

RESULTS

The structure of the bifunctional conjugate, designated EGF-PEG3400-biotin/SA-OX26, is illustrated in Fig. 1A, which depicts thebifunctionality of the conjugate. The two domains of the conjugate,

the tumor EGFR binding domain and the BBB TfR binding domain,are separated by a.200-atom PEG3400 linker. The linker domain isfurther comprised of a biotin attached to the tip of the PEG moiety andSA, which is conjugated to the OX26 anti-rat TfR MAb via a stablethiol-ether bond. A radionuclide domain is attached to human EGFand is comprised of111In chelated to DTPA, which is conjugated toan EGF lysine.

The conjugate retains high-affinity binding to the human EGFreceptor on U87 glioma cells, and this was initially investigated in cellculture as shown in Fig. 1,B and C. There was time-dependentbinding of [111In]-labeled DTPA-EGF-PEG3400-biotin, which is[111In]-labeled EGF in Fig. 1B. This time-dependent binding wasnearly completely saturated by the inclusion of unlabeled human EGF(Fig. 1B). When the radiolabeled EGF was conjugated to OX26/SA toform the overall construct shown in Fig. 1A, there was similarly atime-dependent increase in binding of the conjugate to the U87 cells,and this was inhibited by unlabeled EGF but was not inhibited byunlabeled and unconjugated OX26 MAb (Fig. 1C). The abundantexpression of the human EGFR on the U87 cells in cell culture isshown in Fig. 1D,which is an immunocytochemical study using amurine monoclonal antibody to the human EGFR. The basolateralpattern of the immunostaining is consistent with the expression of theEGFR on the plasma membrane of the U87 glioma cells.

The sustained expression of the immunoreactive human EGFR inthe U87 cellsin vivo in the form of experimental brain tumors in nuderats was demonstrated with immunocytochemistry as shown in Fig.2A for large tumors and in Fig. 2C for small tumors. When [111In]-labeled EGF was conjugated to OX26/SA and injected i.v. into thenude rats with U87 gliomas, the tumors were imaged in the case ofboth large tumors (Fig. 2B) and small brain tumors (Fig. 2D). Con-versely, the brain tumor uptake of the [111In]-labeled EGF, which wasnot conjugated to the BBB delivery system, was negligible (Fig. 2,EandF). Quantitation of the integrated density over same square areaof tumor or normal brain was performed with NIH Image (see “Ma-terials and Methods”). The integrated densities over the tumor andnormal brain were 10796 15 and 556 9, respectively, for studieswith the OX26-EGF conjugate (Fig. 2B). Conversely, the integrateddensity over the tumor or normal brain was not different from thebackground integrated density, 0.496 1.53, for studies with theunconjugated EGF (Fig. 2F).

DISCUSSION

These studies describe an EGF peptide radiopharmaceutical thatis conjugated to a BBB drug targeting system to enable transport ofthe EGF through the BBB in an experimental human U87 glioma.The EGF peptide radiopharmaceutical that is conjugated to theBBB targeting system successfully images small and large braintumors (Fig. 2,B andD), but the unconjugated EGF radiopharma-ceutical does not image the tumors (Fig. 2F), because of lack oftransport of the unconjugated peptide EGF through the BBBinvivo. The conjugate is a bifunctional molecule (Fig. 1A). The EGFpart of the conjugate binds the human EGFR and targets the humanglioma cell, and the OX26 MAb part of the conjugate binds the ratTfR and targets the rat brain capillary endothelium, perfusing thetumor and forming the tumor BBB. The OX26 MAb part of theconjugate enables transport through the BBB, and the EGF part ofthe conjugate enables specific binding to the glioma. The EGF doesnot bind normal brain because of the paucity of EGFRs in normalbrain, and the OX26 MAb does not bind the tumor because theOX26 MAb is specific for the rat TfR and does not bind the humanTfR expressed on the human glioma cells. The bifunctionality ofthe conjugate is retained by the use of the extended PEG3400linker,

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which physically separates the EGF and OX26 halves of theconjugate and enables binding of the conjugate to both the EGFRon the tumor cell and the TfR on the BBB of the tumor (15).

The EGF peptide radiopharmaceutical conjugated to the anti-TfRMAb (Fig. 1A) was used in previous studies to image experimentalbrain tumors in Fischer ratsin vivo (12). These animals had beenimplanted in the caudate putamen with C6 rat glioma cells that hadbeen transfected previously with a gene encoding the human EGFR(18). This gene construct was under the influence of a glucocorticoidinducible promoter. The C6 glioma cells expressed the EGFR in tissueculture in the presence of 1mM dexamethasone but did not express thehuman EGFR when grownin vivo in the form of experimental braintumors (12). These previous studies demonstrated that EGF does notcross the BBB, and that EGF can be made transportable through theBBB by conjugation of the peptide to the BBB delivery system.However, successfulin vivo imaging of brain tumors was not possiblein previous studies because the human EGFR was not expressed onthe C6 glioma cellsin vivo (12). These previous studies showed thatthe expression of the EGFR on the tumor cellsin vivo is a necessarycondition for tumor imagingin vivo, and that tumor imaging does notarise from the uptake of the OX26 MAb conjugate nonspecifically byFc receptors present on the tumor cell. Additional evidence for thespecificity of the conjugate binding to the tumor cell EGFR is theradioreceptor assay, showing inhibition of conjugate binding to theU87 glioma in the presence of excess EGF but not excess OX26 MAb

(Fig. 1C). The OX26 MAb is specific for the rat TfR and does notbind the human TfR on the U87 glioma cells. Rather, the OX26 MAbbinds the rat TfR expressed on the capillary endothelium, perfusingthe tumor and comprising the BBB of the tumor.

The present studies use human U87 glioma cells, which express thehuman EGFRin vivo in experimental tumors in nude mice (17).Because the OX26 BBB drug delivery system is specific for rats andnot mice,4 U87 experimental tumors were generated in the presentstudies in nude rats. Prior investigations have shown that nude ratsmay be used for experimental U87 gliomas (19).

In vivo imaging of brain tumors requires that the radiopharmaceu-tical be labeled with a stable radionuclide. In the present formulation,the radionuclide,111In, was chelated to human EGF via a DTPAlinker, which was attached to one of the lysine moieties on the humanEGF (12). A greater metabolic stability of the EGF radiopharmaceu-tical in vivo is achieved using the111In radionuclide, as opposed to a125I radionuclide (12). The SA was attached to the OX26 MAb via astable thiol-ether linkage. The SA bound the biotin moiety at the tipof the PEG3400 strand, which in turn was conjugated to anotherinternal lysine residue on the human EGF. The structure of the humanEGF after the dual conjugation of DTPA and PEG3400-biotin wasconfirmed in previous studies (12) with matrix-assisted laser desorp-

4 Unpublished observations.

Fig. 1. A, the EGF peptide radiopharmaceutical conjugate is formed by three domains: (i) a glioma-binding domain consisting of EGF radiolabeled stably with111In through themetal chelator, DTPA; (ii) a linker domain consisting of a single strand of PEG3400spacer arm attached to EGF, and a biotin moiety, which is in turn bound to SA; (iii) a BBB transportdomain consisting of the OX26 anti-TfR MAb, which is conjugated to SA through a stable thiol-ether linkage. The OX26 MAb undergoes receptor-mediated transcytosis through theBBB via the endogenous TfR on the capillary endothelium of the tumor, which originates from preexisting rat brain microvessels (13, 14). The EGF moiety binds to the human EGFR,which is expressed on the glioma cells. The PEG3400 linker releases steric hindrance of the OX26 MAb on binding of the EGF to its cognate receptor (15). The111In-labeledradionucleotide is suitable for imaging and confers metabolic stability on the conjugate (12), as compared with the use of other radionuclides such as125I. B, the radioreceptor assayshows the time course of binding of [111In]-labeled DTPA-EGF-PEG3400-biotin ([ 111In]-EGF) to U87 human glioma cells in either the absence (F) or presence (‚) of 1 mM of unlabeledEGF. Each point represents the mean of three wells per point;bars,SE.C, the binding of the conjugate to U87 glioma cells in cell culture is shown in the presence of no additive (F),1 mM unlabeled EGF (‚), or 100mg/ml unlabeled and unconjugated OX26 MAb (E). The structure of the conjugate is shown inA, and the conjugate is[ 111In]-EGF1OX26/SA.D,immunocytochemistry of cultured U87 cells fixed in 100% cold methanol and immunostained with a mouse monoclonal antibody to the human EGFR shows abundant immunoreactivityon the plasma membranes of the U87 glioma cells in cell culture.

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tion ionization mass spectrometry (12). The [111In]-labeled DTPA-EGF-PEG3400-biotin/SA-OX26 is shown in Fig. 1A. Previous studieshave characterized: (a) the structure of the conjugate; (b) the dualbinding of the conjugate to the EGF and transferrin receptors; (c) thein vivo plasma pharmacokinetics of the conjugate; and (d) the meta-bolic stability of the conjugatein vivo (12).

The lack of significant transport of the unconjugated [111In]-labeledEGF into the U87 human gliomas shown in Fig. 2F parallels theabsence of [111In]-labeled EGF entry into C6 gliomas in Fischer ratsin vivo reported previously (12). Although the BBB is disrupted inbrain tumors including U87 experimental brain tumors (20), thedisruption of the BBB is not sufficient to enable imaging of the braintumor with unconjugated peptide radiopharmaceuticals such as EGF.Successful tumor imaging requires the conjugation of the peptideradiopharmaceutical to a BBB drug delivery system (Fig. 1A). Thetumor imaging is not derived from the coadministration of unlabeledEGF, which is given to inhibit peripheral degradation of the EGFconjugate (12). EGF does not cross the BBB (12), and the coadmin-istration of unlabeled EGF with labeled EGF does not increase brainuptake of EGF.4

Once the EGF peptide radiopharmaceutical is delivered through theBBB via the endothelial TfR, the EGF is bound to the tumor EGFR.The sustained binding of the peptide to the EGFR may be attributableto the biological characteristics of EGF binding to its receptor. The

human EGFR binds both EGF and transforming growth factora.However, once internalized, transforming growth factora is rapidlydissociated from the EGFR, whereas EGF remains bound to the EGFRafter internalization (21). This property of EGF binding to its cognatereceptor may underlie the high signal of the experimental brain tumorseen in Fig. 2B. Another aspect of EGFR biology that may enhancethe tumor image in patients with brain tumors is the up-regulation ofthe EGFR on human glioma cells exposed to radiation therapy (22).However, EGF peptide radiopharmaceuticals cannot access the EGFRon brain tumor cells, which are localized behind the BBB, if a BBBdrug delivery system is not used in humans.

The present studies demonstrate that peptide radiopharmaceuticalssuch as EGF can be used to image brain tumors when the moleculesare conjugated to a BBB drug delivery system. Brain tumors could beimaged with either a peptide radiopharmaceutical,e.g., EGF, or anantibody radiopharmaceutical,e.g., an anti-EGFR MAb. Previousinvestigations have attempted to use a radiolabeled anti-EGFR MAbto image human brain grade 3–4 gliomas (9, 23), but tumor uptake ofnonspecific antibodies was also observed in these patients, indicatingthat the tumor was advanced with a fully disrupted BBB (9). How-ever, specific imaging of the tumor in the early phase of the tumorgrowth when the BBB is intact is probably not possible unless thepeptide or antibody radiopharmaceutical is conjugated to a specificBBB drug targeting system such as that used in the present studies.

Fig. 2.A, C,andE, experimental U87 brain tumors were grown in nude rats for 16 days. The brain was removed, and frozen sections were immunostained with a mouse monoclonalantibody to the human EGFR and a secondary antibody comprised of a biotinylated horse anti-mouse IgG antibody that had been preabsorbed with rat immunoglobulin. Theimmunocytochemical study shows high expression of the immunoreactive human EGFR in the brain of nude rats with either large (A) or small (C) U87 gliomas.B, D, andF, filmautoradiography of frozen brain sections obtained from U87 tumor-bearing nude rats injected i.v. with 100mCi of either [111In]-labeled DTPA-EGF-PEG3400-biotin conjugated toOX26/SA (BandD) or [111In]-labeled DTPA-EGF-PEG3400-biotin without conjugation to the BBB delivery system (F). The film autoradiogram shows increased uptake of the EGFpeptide radiopharmaceutical conjugated to OX26/SA in both large (B) and small (D) U87 tumors but negligible brain tumor uptake of the EGF that was not conjugated to the BBBdelivery system (F). The scans inB, D, andF are serial sections from the same 1–2 mm coronal slabs. The immunocytochemistry and QAR images inA-B, C-D,andE-F are fromthe same animal, but the sections were taken from different 1–2-mm coronal slabs of the tumor, which accounts for the small differences between the tumor volume seen withimmunocytochemistry (left panels)versusthe QAR (right panels).BRAIN SCAN IN LIVING ANIMALS,radiolabeled OX26-EGF conjugate (B andD) or unconjugated EGF (F) wasadministeredin vivo, and the frozen sections were subsequently developed by QAR, as opposed toin vitro QAR, where the labeled peptide is applied to tissue sectionsin vitro.

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Although the OX26 MAb is specific for rats, similar studies can alsobe performed in humans using BBB transport vectors that bind tohuman BBB receptors. A MAb to the human insulin receptor is activein humans and Old World primates (24), such as the Rhesus monkey,and has a BBB transport coefficient 9-fold greater than that foundwith anti-TfR MAbs (25). In addition to neuroimaging brain tumors,the use of a BBB drug delivery system and a peptide pharmaceuticalcould also be directed toward the therapy of human brain tumors.Potato carboxypeptidase inhibitor, a T-knot protein, is an EGFRantagonist that blocks cell growth when the peptide is bound to thetumor EGFR (26). Virtually any neurodiagnostic or neurotherapeuticagent can be conjugated to the BBB drug delivery system for nonin-vasive brain drug deliveryin vivo (27).

ACKNOWLEDGMENTS

Daniel Jeong skillfully prepared the manuscript, and Margarita Tayagprovided excellent technical assistance. Dr. Harry Vinters aided in the lowmagnification photography of the brain immunocytochemistry specimens.

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TARGETING EPIDERMAL GROWTH FACTOR RECEPTOR IN BRAIN TUMORS

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