[CANCER RESEARCH 55. 3140-3148, July 15, 1995]

Monoclonal Antibodies against EGFRvIII Are Tumor Specific and React withBreast and Lung Carcinomas and Malignant (¿liornas1

Carol J. Wikstrand, Laura P. Hale, Surinder K. Batra, M. Leslie Hill, Peter A. Humphrey, Shekar N. Kurpad,Roger E. McLendon, David Moscatello, Charles N. Pegram, Craig J. Reist, S. Thomas Traweek, Albert J. Wong,Michael R. Zalutsky, and Darell D. Bigner2

Departments of Pathology ¡C.J. W., L P. H.. S. K. B., M. L H.. S. N. K., R. E. M. C. N. P.. C. J. R.. S. T. T.. D. D. B.] and Radiology ¡M.R. Z.I and the Preuss Laboratory forBrain Tumor Research ID. D. B.l. Duke University Medical Center. Durham, North Carolina 27710; Department of Pathology, Division of Surgical Pathology, WashingtonUniversity Medical Center, Barnes Hospital, St. Louis. Missouri 63110 ¡P.A. HJ: and Jefferson Cancer Institute. Philadelphia. Pennsylvania IVIÜ7ID. M., A. J. WJ

ABSTRACT

Despite molecular biological advances in understanding humancancers, translation into therapy has been less forthcoming; targetingneoplastic cells still requires that tumor-specific markers, preferably those

on the cell surface, be identified. The epidermal growth factor receptor(EGFR) exists in a deletion-mutant form, EGFRvIII, which has been

identified by genetic and immunological means in a subset of gliomas andnon-small cell lung carcinomas. Specific polyvalent antisera to the extra

cellular portion of the variant were readily induced, but immunizationusing a synthetic linear peptide representing the unique EGFRvIII primary sequence has been unsuccessful in mice or macaques. We reporthere five specific monoclonal antibodies (mAbs) developed through long-term immunization protocols using the EGFRvIII-specific synthetic pep-

tide and the intact variant in different formats that maintained secondaryand tertiary conformation. These mAbs identify the EGFRvIII on the cellsurface with relatively high affinity (A'Arange, 0.13 to 2.5 x 10' M~') by

live cell Scatchard analysis. These mAbs are specific for EGFRvIII asdetermined by RIA, ELISA, Western blot, analytical flow cytometry,autophosphorylation, and immunohistochemistry. Isolating specific mAbsenabled us to analyze normal and neoplastic human tissue and establishthat EGFRvIII is truly tumor specific for subsets of breast carcinomasand for previously reported non-small cell lung carcinomas and gliomas.

Also, this receptor is not expressed by any normal human tissues thusfar examined, including elements of the peripheral, central nervous, andlymphoid systems. With mAbs, we identified a higher incidence of EGFRvIII posit i\ il \ in gliomas than previously described and identified anEGFRvIII-positive subset of breast tumors; also, we observed that the

EGFRvIII epitope is not expressed in normal tissues, and we demonstrated the localizing and therapeutic potential of the mAbs for tumorsexpressing this epitope. Our observations strongly warrant developmentof this mAb-antigen system as therapy for breast, lung, and central

nervous system tumors.

INTRODUCTION

The EGFR3 gene (c-erbB-l) is often amplified and overex-

pressed in malignant human gliomas (1, 2). Frequently, thisamplification is correlated with structural rearrangement of thegene, resulting in in-frame deletion-mutant extracellular domainsof the EGFR protein (3-6). One class of deletion-mutant identifiedin some malignant gliomas and non-small cell lung carcinomas(5, 7, 8), EGFRvIII, is characterized by an 801-bp in-frame dele

tion with generation of a glycine residue at the fusion point.Xenografts D256 MG and D270 MG maintain expression ofEGFRvIII (2, 9). The deletion removes NH2-terminal amino acid

Received 2/2/95; accepted 5/15/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this faci.

1This work was supported in part by NIH Grants CA 11898, CA 56115. CA 32672.

NS 29955. and NS 21X123and by American Cancer Society Grant 89-171.2 To whom requests for reprints should be addressed, at Duke University Medical

Center, Pathology Department, Box 3156, Durham, NC 27710.1The abbreviations used are: EGFR, epidermal growth factor receptor; DPBS.

Dulbecco's PBS; IRF, imrnunorcactive fraction; RT-PCR. reverse transcription-PCR.

residues 6 through 273 from the extracellular domain of the intact(Mr 170,000) EGFR, resulting in a M, 145,000 EGFRvIII that hasa unique primary sequence represented by an inserted glycineresidue at position 6 between amino acid residues 5 and 274.

Immunization with short synthetic peptides derived from hydro-philic sequences of human EGFR induces specific anti-EGFR antisera

(10), and we produced polyvalent sera to the EGFRvIII using synthetic peptide immunization. A 14-amino acid peptide correspondingto the fusion junction (Pep 3; amino terminus residues 1-5, gly,residues 274-280, and a terminal cysteine) was chemically synthe

sized, coupled to keyhole limpet hemocyanin, and used to immunizerabbits, a goat, macaques, rats, and mice (11). The resultant anti-Pep3 site-specific antibodies raised in rabbits and the single goat (7, 11)

were highly selective for the parent molecule EGFRvIII, as opposedto intact EGFR as determined by immunoprecipitation of solubilizedmembrane preparations and indirect immunohistochemistry of frozensections of human glioblastoma and derived xenografts. Pep 3 immunization alone was not sufficient to induce significant levels of antibody reactivity against the EGFRvIII in mice, rats, or macaques (11).Isolation of mAbs specific for EGFRvIII were achieved onlyafter immunization protocols were used that combined Pep 3 andEGFRvIII-positive cell or cell membrane immunization. Three mod-erate-to-high affinity anti-EGFRvIII mAbs have rapidly extended our

analysis of normal and neoplastic human tissue. No normal humantissues tested, including those from the peripheral and central nervoussystem, the lymphoid system, skin, breast, liver, lung, ovary, placenta,endometrium, testes, and colon, have been found to express EGFRvIIIby immunohistochemical and/or genetic analyses. The anti-EGFRvIII

mAbs herein, evaluated with immunohistochemical techniques, detected a higher incidence of EGFRvIII expression in gliomas (52%)than was previously reported. For the first time, we demonstrated that8 of 11 (73%) of breast carcinoma cases contain EGFRvIII mRNA, asdetected by RT-PCR followed by ethidium bromide staining. Of these

eight, at least three were immunohistochemically reactive with theEGFRvIII mAb L8A4. This report summarizes the characterization ofthese novel anti-EGFRvIII mAbs and establishes the potential for

their application in multiple immunotherapeutic approaches to a variety of cancers, including those of the breast, lung, and centralnervous system.

MATERIALS AND METHODS

Control and Standard mAb Reagents. As previously described (2, 7, 11),mAb 528 (Ab-1; Oncogene Science, Manhasset, NY) is reactive with the

external domain of intact EGFR, inhibits the binding of EOF, and has beendemonstrated to bind EGFRvIII. Anti-EGFR mAb Z025 (IgGl; Zymed,

Inc., San Francisco, CA) reacts with the cytoplasmic domain of EGFR andEGFRvIII. Anti-EGFR mAb EGFR1 (IgG2b; Boehringer-Mannheim, Indian

apolis, IN) reacts with the external domain of intact EGFR.Cell Lines and Xenografts. Cell line A431 and derived xenografts have

been described previously (2, 9). A431 is considered the reference source bothin culture and in xenograft form of the intact normal human EGFR. Malignant

3140

Research. on September 20, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

SPECIFIC mAbs TO EGFRvIH

gliomas D-256 MG and D-270 MG were simultaneously established in tissue

culture and murine xenograft form as described previously (2, 7, 9); bothxenografts express a M, 145,000 receptor precipitable by mAb 528 and distinctfrom the M, 170,000 receptor precipitated from A431 xenografts by mAb 528.For analysis of xenograft cells, disaggregation of freshly dissected xenograftswas performed in a trypsinization flask in 0.8% collagenase-PBS for 35 min,

followed by filtration through sterile nylon mesh and separation of viable cellsin lymphocyte separation medium (Organon Teknika, Treyburn, NC). Thesepopulations were immediately used in indirect immunofluorescence assays asdescribed below. Cell line HC2 20 d2 was obtained by transfection of NIH 3T3cells with a cDNA corresponding to the same 801-bp in-frame deletion as thatin D-256 MG and D-270 MG (6). Xenografts of the HC2 20 d2 cell line wereestablished in nude mice by the inoculation of 1 X IO8 cultured cells/recipient.

Samples of neoplastic and normal human tissue were obtained through theTissue Bank of the Duke University Comprehensive Cancer Center or throughthe Department of Pathology, Duke University Medical Center.

Microsomal Membrane Preparation. All procedures were performed at4°C;10 g of HC2 20 d2 or A431 athymic mouse xenografts were homogenized

in 20 mM Tris buffer (pH 7.4; 0.3 M sucrose, and 1 mM phenylmethylsulfonylfluoride) and centrifuged (15,000 X g; 20 min), and the pellet (pi) was saved.The supernatant was ultracentrifuged (150,000 X g; 30 min), and the pellet

was washed by ultracentrifugation until the supernatant was free of protein.The final pellet was resuspended in 1 ml of 115 mM phosphate buffer/g oftissue homogenized and stored at -135°C.

Solubilization of EGFR. The pellet (pi) was homogenized in 115 mMsodium phosphate buffer (pH 7.4; 1% Triton X-100, 10% glycerol, and 1 mMphenylmethylsulfonyl fluoride), rocked for 4 h at 4°C,and ultracentrifuged

(150,000 X g; 30 min); the resulting supernatant was assayed for protein andstored at -135°C.

Immunogens, Immunization Protocols, and Fusions. Pep 3, a 14-amino

acid peptide corresponding to the predicted amino acid sequence at the fusionjunction (LEEKKGNYVVTDHC), was synthesized, purified, and coupled tokeyhole limpet hemocyanin by AnaSpec, Inc. (San Jose, CA; Ref. 12). A10-amino acid peptide of unrelated structure (CNLLEGCTGP), Pep 1, served

as negative control. Structural characterization and purity were determined byamino acid analysis and mass spectroscopy at AnaSpec, Inc. and the Macro-

molecular Structure Laboratory of the Duke Comprehensive Cancer Center (7).Four combination immunization protocols, as detailed in Table 1, used the

following immunogens: Pep 3 conjugated to keyhole limpet hemocyanin in a1:1 emulsion in DPBS with complete Freund's adjuvant (Difco, Detroit, MI),incomplete Freund's adjuvant, or in DPBS alone; collagenase-disaggregated

D-270 MG xenograft cells (D-270 MG-X); cultured HC2 20 d2 cells harvestedwith 0.02% EDTA-DPBS; and microsomal membrane preparations of HC2 20

d2 xenograft cells. BALB/c female mice (Charles River Breeding Laboratories, Stoneridge, NY), 8 to 15 weeks of age at the initiation of immunization,were used. In general, reciprocal 50% end point titers in excess of 5000 versusPep 3 and the receptor target were required before fusion.

Table 1 Ami-ECFRvlli mAbs

mAbImmunization regimen" obtained Ig class

Protocol 1 Days 1, 157; Pep 3-KLH J2B9 IgGlDays 56, 132; D-270 MG-X cells J3F6 IgGlDay 161; fusion

Protocol 2 Days 40, 103; Pep 3-KLH L8A4 IgGl

Days 1, 25, 74, 87; HC2 20 d2 cellsDay 107; fusion

Protocol 3 Day 199; Pep 3-KLH Y10 IgG2a

Days 1,213; HC2 20 d2 cellsDays 161, 175; HC2 20 d2 microsomal membranesDay 216; fusion

Protocol 4 Day 68; Pep 3-KLH H10 IgGlDay 1; HC2 20 d2 microsomal membranes Hll IgGlDays 83, 177, 194; Pep 3-KLH + HC2 20 d2

microsomal membranesDay 197; fusion

" Initial immunizations with Pep 3-KLH were in complete Freund's adjuvant; subsequent immunizations were in incomplete Freund's adjuvant. Doses of cells administeredi.p. were 5-10 X IO6 cells. Microsomal membrane preparations were given in either IX,2X, or 25X doses, where IX represents microsomal membranes equivalent to 5 X IO6

cells. KLH, keyhole lympet hemocyanin.

Fusions were performed with the nonimmunoglobulin-secreting Kearney

variant of P3X63/Ag8.653 using our standard procedure (13, 14). Supernatantswere screened for positivity on Pep 3 and D-270 MG-X or HC2 20 d2 and

for lack of reactivity for non-transfected NIH 3T3 cells and A431 (normal

EGFR). Hybrids derived from Protocol 4 were initially screened on HC2 20 d2extract preparation for positivity and A431 extract preparation to determine

specificity.Antibody Purification. Rabbit anti-Pep 3 polyvalent serum and mAbs

were purified as described previously (7, 11, 14, 15), except that mAb L8A4was purified on a Protein G column (GammaBind Plus; Pharmacia).

Assays for Antibody Activity. Antibody titers against plated peptideswere determined by ELISA and RIA (11). A capture ELISA assay using,sequentially, sheep anti-EGFR intracellular domain antiserum (Life Technol

ogies, Grand Island, NY) as capture reagent, antigen extract, prospectiveanti-EGFRvIII supernatants, and sheep antimouse IgG Fc was used to screen

Protocol 4 hybridomas. RIA was used to determine reactivity against cell linesexpressing EGFRvIH (14). A modified Scatchard analysis was used to measurethe binding affinity of lodogen-catalyzed iodinated mAbs (15), beginning with

serially diluted radiolabeled antibody at 10 ng/ml versus HC2 20 d2 and NIH3T3 cells. Data were analyzed using the Equilibrium Binding Data AnalysisProgram (Biomedicai Computing Technology Information Center, Nashville,

TN; Ref. 16). Ascertainment of recovered cells at the end of the procedureallowed calculation of the number of EGFRvIH sites/cell. Iodinated anti-

EGFRvIII mAbs were also analyzed by competitive binding assay; 50 ng ofeach iodinated mAb was reacted with acetone-fixed HC2 20 d2 cells in thepresence of increasing amounts of cold, competing anti-EGFRvIII mAbs orisotype controls to 1000-fold excess (50 fig/ml). After being incubated at 37°C

for 2 h, plates were washed, and 125I counts bound/well were determined.

Similarly, competitive binding inhibition analysis versus Pep 3 was performedby surface plasmon resistance (see description below). Conditions for

inhibition assay included iodinated mAbs at 300 ng/ml versus increasingconcentrations of competing cold mAbs to 166-fold excess (50 fig/ml).

Immunoreactive Fraction Determination. Determination of the IRF ofradioiodinated mAbs using the lodogen method was performed by Lindmoanalysis (17) with HC2 20 d2 xenograft (positive) and athymic rat normal brain(negative) homogenates as targets in an 18- to 24-h assay at 4°C.Plotting the

total divided by the specifically bound activity versus the reciprocal of theantigen concentration yielded a linear plot, the intercept of which represents

the inverse of the IRF.Immunofluorescence and Immunohistochemistry. Analytical flow cy-

tometry was performed with cell populations as described by Bjerkvig et al.

(18), except that cells were harvested by 0.02% EDTA and analyzed in ZincOption-10% PCS on a Becton Dickinson FacSort equipped with Lysys software (Becton Dickinson, San Jose, CA). Assays were performed at 4°C.All

washes were performed with iced medium, and tubes were kept in the darkafter application of the FITC-conjugated secondary reagent. The anti-EGFR

control mAb used (528) recognizes an epitope on the extracellular domain ofwild-type and variant EGFRvIH. The conditions of the assay and reagents used

were chosen to demonstrate cell surface epitope detection. Cells so treatedwere either subjected to analytical flow cytometry or photographed. Immuno-histochemical analysis of acetone-fixed (-70°C, 30 s), 5- to 8-f¿mtissue

sections of human normal or tumor tissue, rat glioblastoma xenografts derivedfrom human tumors or transfected cell lines, or cultured cells plated on LabTekslides was performed as described previously (2, 7).

Immunoprecipitation of EGFR. Solubilization of tissues for immunopre-

cipitation was performed as described previously (2, 19) with some modifications. Three hundred mg of A431, D-270 MG, or HC2 20 d2 xenograft tissue

was homogenized in Solubilization buffer [20 mM HEPES (pH 7.4), 150 mMNaCl, 1% Triton X-100, 10% glycerol, 4 mm iodoacetate, and 1 mg/ml

aprotinin].Immunoprecipitation was performed with Pep 3 affinity-purified polyvalent

rabbit or goat anti-Pep 3, mAb 528, or experimental mAbs. mAb 528 (2 /ig),polyvalent anti-Pep 3 sera (1 pig), or each purified mAb of unknown specificity

(20 (ig) in 115 mM phosphate buffer (pH 7.4) was combined with 2 mg eachof Protein A and Protein G (Sigma) and 200 /¿Iof Protein G+ (PierceChemical Co., Rockford, IL). Immunoprecipitation was performed with a25-mg equivalent of solubilized xenograft tissue with 100 fi\ of the immobilized mAb (antibody-Proteins A, G, and G+ pellet resuspension).

3141

Research. on September 20, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

SPECIFIC mAbs TO EOFRvIII

Autophosphorylation of the EGFR. Washed pellets were resuspended insolubilization buffer plus 2 mM MnCU and 3 ¿iCiof [y^PJATP (ICN; >4()00

Ci/mmol) and prepared for SDS-PAGE analysis as described previously (2).Immunoprecipilates were run on 7.5% SDS-PAGE gels (20) using theMini-Protean II System (Bio-Rad Laboratories, Richmond, CA) at 200 V for40-50 min.

Western Blotting. Western blot analysis was performed as described previously (20); exceptions involved the solubilization of cell pellets of HC2 20d2, A431, and NIH 3T3 cells, the use of an irrelevant lgG2a as negative controlfor Y10, and the use of the enhanced DAB Substrate Kit (Pierce) for development. Concentration of primary reagents for immunostain were 20 fig/ml forall mAbs, with the exception of the anticytoplasmic domain MAb ZO25, whichwas used at 2 fig/ml.

Determination of Affinity of Anti-EGFRvIII mAbs for Pep 3. Kinetic

constants for binding of mAbs to Pep 3 were measured using the PharmaciaBIAcore machine. The coupling kit and protocol provided by the manufacturer(21) were used to immobilize Pep 3 on the biosensor chip, followed byinjection of either a 250-^ig/ml or a 2-mg/ml solution of Pep 3 (0.1 m citrate

buffer, pH 4.0) and blocking of free sites on the sensor chip with 50 mMcysteine/NaCl. Apparent rate constants were determined by injecting a seriesof dilutions in the range of 30 to 2000 nM of each mAb over immobilized Pep3 at a flow rate of 10 /¿l/minin binding buffer [10 mm HEPES (pH 7.4), 150mm NaCl, 3.4 mm EDTA, 0.05% BIAcore surfactant: Pharmacia). Resultantsensorgrams were analyzed via the nonlinear curve-fitting software developed

by Pharmacia (22, 23).Identification of EGFRvIH mRNA by RT-PCR. RNA was purified from

2 x 20-fun sections of frozen breast carcinoma or control tissues using theguanidinium isothiocyanate-acid phenol method (24). Tissue controls includedD-256 MG and the NR6 cell line (25) transfected with the normal EGFR gene

expressing normal EGFR (NR6W) grown as s.c. xenografts in athymic rats andthe cell lines NR6, NR6W. and NR6M, which express no EGFR, normalEGFR, and EGFRvIH, respectively (26). Three ¿tgof total RNA was combinedwith 100 ng random hexamer primers (GIBCO-BRL. Gaithersburg, MD) andRNasin (Promega, Madison, WI); the solution was heated at 68°Cfor 10 min,

then placed on ice. DTT (0.1 M), dNTPs (10 mM each), Superscript reversetranscriptasc (GIBCO-BRL), 5X Superscript buffer, and water were added, andthe mixture was heated at 37"C for 15 min, then 43°Cfor 60 min. The cDNAsynthesis reaction was terminated by heating at 98°C,and the mixture wasstored at -80°C. PCR was performed using 2 jul of cDNA in a total reaction

volume of 75 fil containing 2.5 units Taq DNA polymerase (Promega, Madison, WI); Taq buffer containing 1.5 mM Mgf ~, 0.6 JX.MEGFR forward primer,

and 0.6 /J.MEGFR reverse primer; and 200 /UMdeoxynucleotide triphosphates.A hot start technique was used. Forty cycles of amplification were performed[95°C(80 s), 54°C(1 min), and 72°C(2 min)], and final elongation was

performed for 10 min. A negative control lacking template was run with thereaction. Products were analyzed by electrophoresis on 2.0% agarose gels intriacetate-EDTA buffer (0.02 M Tris-acetate-0.001 M EDTA) using 100-bpmarkers (GIBCO-BRL) as size standards, followed by ethidium bromidestaining. Primers for PCR of wild-type EGFR and/or variants were forward5'-GGGGAATTCGCGATGCGACCCTCCGGG-3' and reverse 5f-GGGAA-GCrriCCGTTACACACrriGCG-3'. Eighteen bases in each primer were

complementary to the nucleotide sequences for human EGFR published previously (27). Each primer also contained an artificially introduced restrictionsite at its 5' end to facilitate the cloning of the resultant PCR products into

pBluescript vector (Stratagene, La Jolla, CA) for sequence analysis. Whenthese primers are used, the sizes of the expected normal and EGFRvIIIproducts are 1037 and 236 bp, respectively.

Sequencing. Double-stranded cDNAs were prepared using standard

techniques (28), and Sequenase bacteriophage T4 DNA polymerase wasprepared under conditions recommended by the supplier (U.S. Biochemi-

cals, Cleveland, OH).

RESULTS

Isolation of Specific Anti-EGFRvIII mAbs. Fusion and screen-

D-270 MG cultured cell line or the nontransfected NIH 3T3 parent

cell line and the normal EGFR overexpressing cell line A431. OneIgG 1 hybrid reactive with A431 and HC2 20 d2 solubilized receptor,Hll, was retained as a control reagent for normal EGFR. As shown inTable 1, five mAbs (four IgGl and an IgG2a) were potentially specificfor EGFRvIII. These were cloned, ascites were induced in athymicmice, and antibody was purified.

The fusion performed after immunization under Protocol 4 (Table1), which yielded mAb H10, allowed the investigation of someparameters of EGFR immunogenicity. Of original outgrowing colonies in single wells, 3 of 35 (8.6%) were nonreactive with targetsEGFR and EGFRvIII (microsomal membranes), 29 of 35 (82.8%)were reactive with both targets, and 3 of 35 (8.6%) were reactive withEGFRvIII microsomal membranes alone. Of the uncloned hybridomassecreting antibody reactive with solubilized EGFR and EGFRvIII,only 7 of 29 (24.1%) were reactive with cell surface receptors onA431 and HC2 20 d2 cells as detected by analytical flow cytometry,indicating that the majority (22; 75.9%) were reactive with epitopescommon to EGFR and EGFRvIII not situated on the extracellulardomain.

Reactivity of Anti-EGFRvIII mAbs for Pep 3 and Target Cells.The five putative anti-EGFRvIH-specific MAbs were tested versus the

linear synthetic peptide, Pep 3, which spans the fusion junction, and

A4,

40 -

Q 32

O 24 -I

m 16 -

g::.,.

10» IO1 10§ 10-

jig/rnl Ig

10-

Ma/ml Ig

Fig. 1. Titration of anti-EGFRvIH mAbs versus immunogens. A, activity versus Pep 3(control, irrelevant 10-mer peptide Pep I). Assay performed as described in "Materialsand Methods"; binding ratios for all mAbs versus irrelevant Pep 1 ranged from 0.6 to 1.6

ing were performed (14) with the specific targets used for each over the tested mAb concentration range of 20 to 0.15 ^g/ml. fi. activity vera«cell-borneimmunization protocol. mAb retention required reactivity for the E™' f exp,ressefd"I,""20 d2 ce"s (negative T'™,',' untranffcd ,f]H ™

1 ' * cells). Binding ratios for all mAbs versus negative control cells ranged from 0.4 to 1.8immunogens and lack of reactivity for the EGFRvIII-nonexpressing over the tested mAb concentration range of 20 to O.OOl

3142

Research. on September 20, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

SPECIFIC mAbs TO EOFRvlll

HC CELLSp 60

A431 CELLSp 140

Hll

IgG

CONTROLS

Fig. 2. Analytical now cytometry of mAbs 528, J2B9, J3Ffi, YIO, H10, L8A4, and HI 1 vera«.!HC2 20 d2 and A431 cells. As IgGl and IgO2a conlrol populalions were essentiallysuperimposahlc, the IgGl control is shown because it controlled for five of seven mAbs tested. A. EGFRvIII transfccted HC2 20 d2 cells (positive target). All mAbs recognize EGFRvlIIon the cell surface (mAbs 528 and Hll. normal EGFR and EGFRvIII; mAbs J2B9, J3F6, YIO, H10. and L8A4, EGFRvIM only). B. A431 cells (normal EGFR only). mAbs 528 andHll define a positive cell population: anti-EGFRvlII mAb reactivity was identical to control IgG reagents.

against the EGFRvIII cDNA transfected cell line HC2 20 d2. Asshown in Fig. \A, the five MAbs differed in their titers to Pep 3 asdetermined by indirect ELISA; 50% end point titers ranged from 1fj.g/ml (mAbs L8A4 and YIO) to 0.05 /j.g/ml (mAb J2B9). Irrelevantisotype controls were negative when tested against Pep 3; the bindingratios for all mAbs versus the unrelated 10-mer Pep 1 ranged from 0.6

to 1.6 over the tested mAb concentration range of 20 to 0.15 /j,g/ml.A similar range of immunoglobulin concentrations was tested inindirect RIA versus acetone-fixed HC2 20 d2 cells (Fig. IB); 50% end

point titers ranged from 5 /ng/ml (mAb J2B9) to 0.8 p,g/ml (mAbsL8A4 and YIO). The lowest titer anti-Pep 3 mAbs (L8A4 and YIO)were the highest titer anti-HC2 20 d2 mAbs in this assay. Irrelevant

controls were negative on HC2 20 d2 cells. The binding ratios for allmAbs versus the untransfected NIH 3T3 cell line ranged from 0.4 to1.8 over the tested mAb concentration range of 20-0.001 fig/ml.

Reactivity of Anti-EGFRvIII mAbs for Various Target Cells.By analytical flow cytometry, putative anti-EGFRvIII-specific mAbs

J2B9, J3F6, YIO, H10, and L8A4 positively stained the surface ofEGFRvIII-positive HC2 20 d2 cells (Fig. 2A) but were unreactive

with A431 cells, which express only normal receptor (Fig. 2B). NIH3T3 cells (the nontransfected control, estimated by iodinated EOFbinding analysis to express approximately 5 X IO3 normal EGFR

molecules) were unreactive with anti-EGFRvlII mAbs (data not

shown). In contrast, mAb Hll, identified during screening as reactivewith A431 microsomal membranes, strongly stained a homogeneouspopulation of A431 cells, similar to that population recognized bymAb 528 (Fig. 23).

Confirmation of Normal versus Variant Receptor Recognitionby mAbs. Confirmation of receptor recognition by the mAbs underinvestigation was demonstrated both by immunoprecipitation withsubsequent autophosphorylation analysis and by Western blotting.Both Pep 3 affinity-purified goat and rabbit anti-Pep 3 sera specifically precipitate a M, 145,000 moiety from D-256 MG and D-270 MG

xenografts, whereas MAb 528 precipitates both the Mr 145,000 molecule from these targets, and the M, 170,000 wild-type EGFR from

A431 cells (7, 11). As shown in Fig. 3/t, mAbs J3F6 and J2B9precipitate an autophosphorylating protein identical to that precipitated by anti-EGFRvlII goat and rabbit antisera. A similar demonstra

tion of specificity is provided by Western blot (Fig. 3B) for antibodiesL8A4, H10, and YIO, as compared with the recognition of both the M,

170,000 and M, 145,000 molecules by mAbs ZO25 and HI 1 in A431cells.

Determination of Affinity of anti-EGFRvlII Antibodies forPep 3. We measured the apparent rate constants of the anti-EGFRvlII

mAbs for the linear peptide immunogen Pep 3 using surface plasmonresonance. This technique allows measurement of real-time biospe-

cific interactions of the apparent rates of association and dissociation(29, 30). Results presented in Table 2 show the apparent on (£„„)andoff (*„„)rates for mAbs L8A4, H10, and YIO. The affinity constant

N V N V N

170

145I

Mob Goat

528 Pep-3

I45k0

Mab

J2B9

Rabbit

8495

BN V N

I7O

145 fV N

|J

V N V N V

ZO25 Hll L8A4 MIO YIO

Fig. 3. Identification of EGFR recognized by mAbs. A. autophosphorylation assay. Immunoprecipitation of either normal EGFR (N) or EGFRvIII (V) by mAbs; comparison withprecipitation by Pep 3 affinity-purified antisera. Source of normal EGFR, A431 xenografttissue; source of EGFRvIII, D-270 MG xenograft tissue. Clear distinction of the M, I70.IXX)normal EGFR i-muv the M, 145,(KK)EGFRvIII is shown. B. Western blot analysis. Immu-nostaining of 7.5% SDS-PAGE (reducing conditions) samples of EGFR and/or EGFRvIII byvarious mAbs. Source of normal EGFR. A431 xenograft tissue; source of EGFRvIII, HC2 20d2 xenograft tissue. mAbs ZO25 and Hll identify both M, 170.1XX)and I45.IXX)bands ofnormal and variant receptor; mAbs L8A4. H10, and YIO identify only EGFRvIII.

3143

Research. on September 20, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

SPECIFIC mAbs TO EGFRvlII

Table 2 Anii-EGFKvlII mAb affinity determination

Range of estimated affinity constants

mAbJ2B9J3F6L8A4HIOY10K(on)(M~ sec)NDND2.3

XIO58.0XIO42.1X IO4PEP

3"K(off)(M~'>NDND6.1

X10~32.2X10~32.4X IO'3KA(M-')NDND3.8

XIO73.6XIO78.5X 10"receptor

'1.6-2.0

X10s1.5-2.4X10s.43-2.5XIO9.59-1.5XIO91.3-2.1X IO8sites/HC2

20 d2cell'8.1-8.8

XIO58.9-9.3X10s2.0-6.3XIO62.1-2.5XIO62.0-5.2X IO6No

ofassays''22433

" Affinity constant determined hy BIAcore analysis versus immobilized Pep 3 as described in "Materials and Methods." (tl)n, on rate; Aroff,off rate; KA, apparent affinity constan!

(kajkofí); ND, not done.Affinity constant determined by Scatchard analysis with iodinated mAb versus HC2 20 d2 cultured cells as described in "Materials and Methods"; calculations were corrected for

IRF determined by Lindmo analysis.' Estimated number of sites on HC2 20 d2 cells determined by assumption of 1:1 stoichiometry of antibody-receptor interaction, using ßmaxfrom the Scatchard plot to determine

total pmol iodinated antibody bound/known number of cells.'' Number of individual assays performed to calculate affinity for cell-associated receptor and number of sites/cell.

3000 -i

2500 -

^ 2000 -

a.WK

1500mo

1000-L8A4

-500

100 200

Time (s)

Fig. 4. BIAcore analysis of anti-Pep 3 affinity. Sensorgram illustrating the binding and

dissociation of mAbs L8A4, H10, and Y10 to Pep 3 on the biosensor chip. mAbs wereinjected at a flow rale of 10 filmili and at a concentration of 250 nM. The flow cellimmobilized with 250 jig/ml of Pep 3 was used for this experiment. Each sensorgram wasadjusted such that the baseline would be 0 resonance units (RU).

(KA = kon/kof{) is also shown. A representative sensorgram, illustrat

ing the differing association and dissociation phases of these mAbs, ispresented in Fig. 4.

Affinity of Anti-EGFRvIII Antibodies for Cell-borne Receptor.

mAbs J3F6, J2B9, L8A4, H10, and Y10 were directly iodinated by thelodogen method; IRF was calculated as described in "Materials andMethods"; and affinity for EGFRvIlI, as expressed on HC2 20 d2

cells, was determined by modified Scatchard analysis, correcting forIRF. The range of IRF values for assays presented in Table 2 were0.35-0.36 (J2B9), 0.42-0.48 (J3F6), 0.68-0.82 (L8A4), 0.89-0.94(H10), and 0.74-0.97 (Y10). Nontransfected NIH 3T3 cells served as

the negative control cell line. As shown in Table 2, mAbs J2B9, J3F6,and Y10 demonstrated similar affinity for cell-borne receptor, rangingin multiple assays from 1.3-2.4 X IO8 M~', whereas mAbs L8A4 andH10 demonstrated affinities in the range of 0.43-2.5 X IO9 M~'. The

number of potential EGFRvlII receptors/cell were calculated by usingthe Scatchard-defined ßmux.These estimates were quite consistent,with a median value from nine assays of 2.1 X IO6 estimated

EGFRvlII receptors/HC2 20 d2 cell. Representative Scatchardplots for mAbs L8A4, H10, and Y10 are presented in Fig. 5.

Fig. 5. Affinity determination of anti-EGFRvIII mAbs L8A4.H10, and Y10 versus transfected HC2 20 d2 cells. All mAbs werelabeled by the lodogen method. Scatchard analysis was correctedfor the immunoreactive fraction of each mAb as determined byLindmo assay (L8A4, 0.676; H10, 0.942; YIO, 0.971).

IllLUCC

Oco

15.0 -

12.0 -

10.5 -

»L8A4:K,„:(1.6 ±0.3) X 109 M"1

# of sites: 3.3 x lO'/cell

coeff. corr.: .93

•H10:Kiff: (5.9 ±1.2) x 108 M"1

# of sites: 2.5 x 10e/cell

coeff. corr.: .92

Y10:K.,f: (1.3 ±0.2) x 108 M'1

# of sites: 2.2 x 106/cell

coeff. corr.: .94

01.0 X IO"«

3144

5.0 x IO'1 1.0 X IO'1

BOUND [MOL]

Research. on September 20, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

SPECIFIC mAbs TO EGFRvIlI

By competitive binding assay to Pep 3, anti-EGFRvIII mAbs L8A4

and H10 were mutually inhibitory, resulting in >90% inhibition incold mAb excess. By competitive binding assay to acetone-fixed HC220 d2 cells, all anti-EGFRvIII mAbs were competitive, resulting in

>92% inhibition in cold MAb excess; irrelevant isotype controlsresulted in s 17% inhibition in 1000-fold excess. The sole exception

was the inability of MAb H10 to inhibit MAb L8A4 binding completely, achieving only 55% inhibition at 1000-fold excess. mAbL8A4, however, completely (97%) inhibited binding of 125IH10.

Immunohistochemical and RT-PCR Analysis of Normal and

Neoplastic Human Tissue. Purified mAbs were screened againstacetone-fixed HC2 20 d2, NIH 3T3, and A431 monolayers or acetone-fixed frozen sections of D-256 MG and D-245 MG human glioma

xenografts passaged in athymic rats. It was determined that mAbsL8A4 and Y10 were the most optimal reagents for immunohistochem-

istry, and they were incorporated into an antibody panel consisting ofPep 3 affinity-purified rabbit antiserum, mAb 528, and mAb 3B4 (panhuman tissue positive control). mAb 528 was chosen for immunohis-

tochemical analysis because it reacts with an epitope common to theextracellular domain of both wild-type EGFR and EGFRvIlI, whilethe most commonly used immunohistochemical anti-wild type EGFR

mAb, EGFR1 (31), has not been demonstrated to bind to EGFRvIlI.Normal IgG or isotype controls included normal rabbit IgG andmurine IgGl and IgG2a at correlative concentrations. As shown inTable 3, tissues examined included 11 cases of breast carcinoma, 31cases of glioma, and a panel of 35 samples of normal tissues. Thetumor types studied were chosen to complement our previous analysisof non-small cell lung carcinomas with Pep 3 affinity-purified polyvalent rabbit anti-Pep 3 antiserum (8). As shown in Table 3, among

the panel of 11 breast tumors, 3 cases (2 infiltrating ductal carcinomaand 1 intraductal carcinoma) were found to be immunoreactive, fo-cally, with both mAb 528 and the anti-EGFRvIII mAbs in contrast to

negative binding with an irrelevant control IgGl.To investigate the expression of EGFRvIlI by breast carcinomas

further, we isolated mRNA from sections of 10 of 11 of the sametissue blocks studied immunohistochemically and analyzed them forexpression of EGFRvIlI using RT-PCR. Results are shown in Fig. 6.

Products corresponding to PCR amplification of EGFRvIlI mRNAwere present in 3 of 3 breast carcinoma tissues that were reactive withL8A4 MAb immunohistochemically (Fig. 6/4, Lanes 4-6), confirm

ing the specificity of mAb L8A4. In addition, bands corresponding toEGFRvIlI were detected in five additional breast carcinomas that haddemonstrated no immunohistochemical reactivity with MAb L8A4(Fig. 60, Lanes 10-11 and 13-15). Therefore, the presence ofEGFRvIlI mRNA was detected by RT-PCR by ethidium bromide

staining in 8 of 10 of the 11 breast carcinoma tissues evaluated byimmunohistochemistry. Two of the eight tissues containingEGFRvIlI coexpressed normal EGFR (Fig. 6, Lanes 10 and 15).EGFRvIlI RT-PCR products (Fig. 6, Lanes 4, 6, and /5) were

further characterized by direct DNA sequencing. The nucleotidesequence (CTG GAG GAA AAG AAA GGT AAT TAT GTG GTGACA GAT CAC) and the corresponding deduced amino acidsequence (Leu-Glu-Lys-Lys-Gly-Asn-Tyr-Val-Val-Thr-Asp-His)

around the fusion junction were exactly the same as those published earlier by our group (7). Our increased detection of EGFRvIlI by RT-PCR, as compared with immunohistochemical analysis,reflects either the greater sensitivity of PCR-based assays or the

lack of translation of existing EGFRvIlI mRNA.Within the panel of gliomas examined (Table 3), MAb 528 was

reactive with ^25% malignant cells in 4 of 7 anaplastic astrocytomasand 18 of 21 glioblastomas (including 2 of 3 gliosarcomas). Incontrast, EGFRvIlI was immunolocalized in >25% malignant cells of

1 of 4 anaplastic astrocytomas and 13 of 21 glioblastomas (including

Table 3 Reactivity- of frozen human tumor and normal tissue sections withanti-EGFR and anli-EGFRvlll mAbs

Positive reactivity

Tumor or normal tissueclassificationBreast

carcinomaInfiltrating ductalIntraductalNo.

ofcases11

101Anti-EGFRmAb

528+R.

%3/11

272/10 201/1Anti-EGFRvIIImAbs

L8A4and Y10"+/2

%3/11

272/10 201/1

GliomaAnaplastic astrocytomaGliosarcomaGlioblastoma multiforme

Normal tissues'"

Colon, kidney, testes, lung,cerebellum, cerebral cortex, liver

Skin, peripheral nerve, lymph node

Ovary, bone marrow

Spleen

31 24/31 77 16/31 527 4/7 57 1/7 143 2/3 66 2/3 66

21 18/21 86 13/21 62

3/3c

2/2*

0/2

4/4^

0/3

0/2

0/2

4//" mAbs L8A4 and Y10 reacted identically with 10 of 21 glioblastomas and 2 of 3

gliosarcomas. mAb L8A4 positively stained an additional three glioblastomas and oneanaplastic astrocytoma; in these latter cases, YK) reactivity was marginal and interpretedas negative.

'' Normal EGFR tissue distribution was verified with mAb EGFR 1 (31)on human skin

and avidin-biotin-blocked liver, where the characteristic epidermal cell layer staining ofskin (31) and hepatocellular staining, accentuated in periportal hepatocytes (31, 32), wasobserved. The staining pattern with mAb 528 was fainter and decorated a subset of cellsidentified with EGFR1 but served as a control for the anti-EGFRvIII mAbs.

' Endothelial cells in all samples of cerebral cortex and cerebellum stained positive

with mAb 528. Focal areas of perivascular staining were also evident in colon. Lightstromal or septal staining was evident in lung and testes with mAb 528 (33).

'' Light staining of Kuppfer cells occurred with all antibodies, despite blocking with

15% normal secondary serum. Because irrelevant subclass controls exhibited the samepattern, this was interpreted as nonspecific staining.

e Macrophage in peripheral nerve and endothelial cells in peripheral nerve and lymph

node were positive with mAb 528.' Immunohistochemically, all four spleens exhibited light, diffuse staining of connec

tive tissue stroma. in B-cell areas around germinal centers and in the red pulp with mAb528, L8A4, and Y10. which was more pronounced than in primary isotype controlsections. Extensive analysis of two of these spleen samples by lysate preparation, SDS-

PAGE and Western blot, and analytical flow cytometry of Lymphocyte SeparationMedium (Organon Teknika, Treyburn, NC) gradient-prepared lymphocytes, and of one ofthese two samples by RT-PCR, failed to yield any evidence of either normal or EGFRvIlI

protein or RNA expression.

2 of 3 gliosarcomas). These results indicate that virtually all (>85%)of gliomas express normal EGFR and that a significant subset expressEGFRvIlI as well.

The cell surface membrane localization of EGFRvIlI is shown inFig. 7. Freshly disaggregated D-256 MG xenograft cells derived from

a human biopsy were prepared for indirect immunofluorescence assayas described. The cells demonstrate distinctive cell surface stainingwith anti-EGFRvIII mAb L8A4 primary reagent (Fig. 7/4), as opposed

to a total lack of staining with irrelevant IgGl isotype control (Fig.IB). The pattern obtained with mAb 528 was identical (data notshown). Normal skin (two cases), breast (three), lung (two), ovary(three), colon (three), kidney (two), endometrium (two), and placenta(five) are unreactive with affinity-purified rabbit anti-Pep 3 serum (8).

In this series, as shown in Table 3, the mAb panel described abovewas applied to additional samples of normal tissues. Again, no reactivity with the anti-EGFRvIII mAbs was detected, whereas expression

of normal EGFR, as detected by mAbs 528 and EGFR1, was confirmed as detailed in Table 3.

DISCUSSION

In an earlier study, we reported that synthetic peptide immunizationto elicit anti-EGFRvIII activity in mice and macaques was not effec-

3145

Research. on September 20, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

SPECIFIC mAbs TO EGFRvlII

B.

Fig. 6. Detection of EGFRvlII mRNA in breastcarcinoma tissues by RT-PCR. mRNA was extracted from tissue sections and subjected to RT- 1037 [,p».

PCR as described. A. Lane I, base pair markers;Lane 2. D-256 MO xenografts (EGFRvlII +); Lane3. NR6W xenograft (EGFR +); Lanes 4-6. breasttumor biopsies positive with mAb L8A4 by immu-nohistochemistry; Lane 7, negative controls lackingtemplate; Lane 8. negative controls lacking reversetranscription. B. Lanes 1-3, 7. and 8, designationssame as for A; Lanes 9-15, 7 of 8 breast tumorbiopsies negative with mAb L8A4 by ¡mmunohis- 236 bp »•

tochemistry.

1037 bp

•236bp

23456 7 8 2 3 9 10 11 12 13 14 15 7 8

live (11). Because the EGFRvIII-expressing cell targets available inthat study expressed low levels of EGFRvlII receptors (5.7 X 10s),

low affinity responses to the intact receptor may not have beensignificantly positive in our screening assay. The production of highaffinity murine anti-EGFRvIII-specific mAbs was achieved by immu

nization with the EGFRvlII molecule, either as a component of theintact cell surface or of microsomal membrane preparations (Table 1).Such an increase or induction of anti-native protein activity following

presentation of the conformational epitope would suggest that theBALB/c murine response to the EGFRvIII-specific epitope in these

systems may be primarily conformational in nature.The reactivity of the anti-EGFRvIII mAbs listed in Table 1 was

established by multiple approaches, but most critically by positivereactivity by ELISA, RIA, immunoprecipitation, and blotting ofEGFRvIII-transfected cells, membrane preparations, or lysates versus

total lack of reactivity for the nontransfected control cells. The lack ofreactivity for normal EGFR as expressed by A431 cells in these assaysestablished specificity for the EGFRvlII.

The mAb immunohistochemical analysis represents confirmationand extension of results with affinity-purified polyvalent rabbit anti-Pep 3 sera (7, 8). mAb 528 was chosen as the anti-EGFR control mAb,

since it binds an extracellular domain epitope expressed on bothnormal EGFR and EGFRvlII. The normal tissue distribution of EGFRdetected in this series was confirmed by use of mAb EGFR1 and isconsistent with results published previously (31-34). Most signifi

cantly, these results reconfirm the absence of EGFRvlII expression byan extended normal human tissue panel. The incidence of EGFRvIII-positive tumors as defined by anti-EGFRvIII mAb reactivity here

(52%) is higher than the estimate reported previously with polyvalentanti-EGFRvIII sera [6 of 27 (22%) gliomas positive; Ref. 7]. Of the 11

Fig. 7. Indirect immunofluorescence analysis of freshly disaggregated human biopsy-derived D-256 MG xenograft cells, a, mAb L8A4 (5 /ig/ml) as primary reagent; b,irrelevant isotype control IgG (5 /ig/ml), as primary reagent. X 2520.

cases common to these two series, 8 previously identified as negativewith the rabbit antiserum were found in this series to be positive withthat reagent and mAbs 528, L8A4, and Y10. It is probable that thisdiscrepancy is due to our demonstration with antigen-positive xe

nograft tissue that optimal staining of EGFRvlII is attained when thefrozen sections are cut <24 h before assay. In the previous study, thisinterval was not maintained. In addition, mAbs 528, L8A4, and Y10are highly consistent in their staining of control tissue on repeatassays, whereas the polyvalent system is more variable in terms ofnonspecific background and staining intensity on frozen tissue. Theseimproved immunohistochemical approaches allowed us to describe,for the first time, the detection of EGFRvlII protein in breast carcinomas. Parallel RT-PCR analysis revealed a combined incidence of 8of 11 (73%) of breast cancer cases with EGFRvlII mRNA or immu-

nohistochemically detectable protein. Western blot analysis has confirmed that a high percentage of breast carcinomas express thisreceptor.4 In some instances, the small amount of histologically in

terpretable tissue rendered immunohistochemical determination ofreactivity difficult, perhaps contributing to the lower incidence byimmunohistochemistry in breast tissue. Investigation of antigen retrieval techniques and further paired analyses of tumor biopsies areunder way to determine the incidence of the EGFRvlII rearrangementand whether or not the nature of EGFRvlII protein expression mayvary with cell type, resulting in variable levels of immunologicaldetectability.

In terms of antibody specificity and affinity, the degree to whichimmunization with the linear EGFRvIII-specific 14-mer Pep 3 con

tributed to the immunological evolution of the hybrids obtained isunknown. As reviewed extensively by Zeder-Lutz et al, (35), studiesof anti-peptide antibody affinity for peptide and cognate protein rarely

report quantitative comparisons of the affinities for peptide and protein. This is due to the difficulty experienced in maintaining theprotein conformational structure in assays that allow rigorous determination of not only equilibrium affinity constants but also rateconstants. In their study of the affinity of 33 mAbs raised againstpeptide 110-135 of the tobacco mosaic virus for both peptide andcognate protein by BIAcore technology, Zeder-Lutz et al. (35) foundthat the 10- to 50-fold lower affinity constants for protein, as com

pared with peptide, were primarily attributable to differences in theassociation constants. Although the anti-Pep 3 rate constants of only

three mAbs were similarly analyzed here, the association constantsexhibited a wider range of variability (10-fold) than did the dissociation rate constants (3-fold); the calculated KA for mAbs L8A4 and

4 D. Moscatello, C. Ramirez, and A. Wong, manuscript in preparation.

3146

Research. on September 20, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

SPECIFIC mAbs TO EOFRvllI

H10 for Pep 3 are very similar (3.6 and 3.8 x IO7 M ', respectively).

Similarly, the estimated functional affinity (avidity) constants forcell-expressed EGFRvIII of these mAbs as determined by modifiedScatchard analysis are also quite similar (0.43-2.5 X 10g M~' formAb L8A4; 0.57-1.5 X IO9 M"' for mAb H10). As discussed by

Kaufman and Jain (36), however, the apparent affinity of a bivalentmAb measured in equilibrium binding experiments, especially Scat-chard analysis, assumes that the association reaction is a single-step

process and that homogeneity in bound antibody valence exists. Asthe bivalent binding of an IgG molecule is dependent upon theconcentration of antibody in solution, the cellular antigen density, andsteric constraints of both antigen and antibody (36), the authorssuggest that the only potential use of bivalent equilibrium bindingconstants as determined in the Scatchard plot would be in antibodyscreening, where experimental conditions, primarily antigen densityand presentation, can be identical for all antibodies. These conditionsare met here as illustrated in Table 2, where antigen concentration isconsistent, and repeat estimations of affinity constant for mAbs L8A4and H10 are highly uniform. Because these values represent thepotential enhancement of avidity afforded by bivalent attachment asopposed to the postulated monovalent binding in BIAcore analysis, itis not possible to conclude here whether the intrinsic affinity for thecognate protein is greater than that for the linear peptide in this study.

Whether the apparent affinity of a potential localizing mAb iscrucial to intratumoral distribution has been the subject of recentdebate. In a series of studies, Fujimori et al. (37, 38) and Van Osdolet al. (39) demonstrated that iodinated mAbs of higher affinity wouldproduce a higher cumulative absorbed dose of radiation near thesurface of a tumor nodule, and that in larger tumors, the morehomogeneous dose distribution obtained with lower affinity mAbswould potentially result in greater cell kill. Similarly, Langmuir et al.(40), in a study of differing affinity mAb penetration of multicellularspheroids, have shown that, despite a 36-fold difference in bindingaffinity, the iodinated lower affinity mAb 17-14 (5.2 X IO7 M~') wasnearly as toxic as iodinated mAb 323/A3 (1.9 X 10g M~') for LS174T

human adenocarcinoma spheroids. If corrections for the reduced binding levels of the former were made, the lower affinity mAb was foundto be far more effective in terms of kill/dose. Because the short-rangeisotope 125Iwas used, the superior effect was attributed to the moreuniform distribution of 125I-labeled 17-14 in the spheroids. Thus, for

therapy that uses a mAb conjugated to a short-range isotope or an

internalized toxin, an antibody of lower affinity may well be moredesirable. Correlatively, a lower density of antigen sites/cell couldpotentially allow more rapid and even distribution of mAb throughoutthe tumor mass, potentiating a wider range of cell kill.

In summary, five specific anti-EGFRvIII mAbs have been generated, and three exhibit sufficient moderate-to-high avidity for cell-

borne receptor to be developed as potential immunolocalizing andimmunotherapeutic agents. The potential incidence of EGFRvIII expression in gliomas established here is higher than in earlier projections (5, 7). With EGFRvIII being readily detectable in breast andnon-small cell lung carcinomas, we believe that the tumor specificity

of this variant receptor with enhanced tumorigenic potential (26, 41)warrants the continued investigation of these mAbs in a variety ofimmunotherapeutic settings. Experiments recently completed in aseparately submitted study have established the successful localization of iodinated mAbs L8A4 and H10 to EGFRvIII-expressing

xenografts in the murine s.c. xenograft model. The maximal levels ofpercentage of injected dose/g obtained in tumor for these mAbsranged from 10-35%. In addition, preliminary data indicate mAbs

L8A4, Y10, and H10, chemically coupled to Pseudomonas exotoxinmolecules, are quite effective, specific cytocidal agents for EGFRvIII-

expressing cells in vitro (42). Also, as recently postulated (43, 44), the

EGFR, and presumably the EGFRvIII, can be the target for mAb-

induced antiproliferative effects via signal transduction requiringfunctional tyrosine kinases. These approaches to immunolocalizationwith intact and fragmented immunoglobulin can now be investigatedfor the treatment of EGFRvIII epitope-expressing tumors. Rapidscreening of potential EGFRvIII-positive populations is now possiblethrough mAb immunohistochemistry and correlated RT-PCR analysis

and could possibly identify a larger potential tumor target populationthan suspected previously.

ACKNOWLEDGMENTS

We express our appreciation to Elizabeth Purvis, Beth Isley, M. KelliJordan, and Paula Greer for expert technical assistance and to Ann Tamariz,Editor in the Life Sciences, for editorial expertise.

REFERENCES

1. Wong, A. J., Bigner, S. H., Bigner, D. D., Kinzler, K. W., Hamilton, S. R., andVogelstein. B. Increased expression of the epidermal growth factor receptor gene inmalignant gliomas is invariably associated with gene amplification. Proc. Nati. Acad.Sci. USA, 84: 6899-6903, 1987.

2. Humphrey, P. A., Wong, A. J.. Vogelstein, B., Friedman, H. S., Werner, M. H.,Bigner, D. D., and Bigner. S. H. Amplification and expression of the epidermalgrowth factor receptor gene in human glioma xenografts. Cancer Res., 48:2231-2238, 1988.

3. Yamazaki, H., Ohba. Y., Tamaoki, N.. and Shibuya. M. A deletion mutation withinthe ligand binding domain is responsible for activation of epidermal growth factorreceptor gene in human brain tumors. Jpn. J. Cancer Res., 81: 773-779. 1990.

4. Sugawa, N.. Ekstrand. A. J., James, C. D., and Collins. V. P. Identical splicing ofaberrant epidermal growth factor receptor transcripts from amplified rearranged genesin human glioblastomas. Proc. Nati. Acad. Sci. USA, 87: 8602-8606, 1990.

5. Ekstrand, A. J., Sugawa. N., James, C. D., and Collins. V. P. Amplified andrearranged epidermal growth factor receptor genes in human glioblastomas revealdeletions of sequences encoding portions of the N- and/or C-terminal tails. Proc. Nail.Acad. Sci. USA, 89: 4309-4313, 1992.

6. Wong, A. J., Ruppert, J. M.. Bigner, S. H., Grzeschik, C. H., Humphrey. P. A.,Bigner, D. D., and Vogelstcin, B. Structural alterations of the epidermal growth factorreceptor gene in human gliomas. Proc. Nati. Acad. Sci. USA, 89: 2965-2969, 1992.

7. Humphrey, P. A., Wong, A. J., Vogelstein. B., Zalutsky. M. R.. Fuller, G. N., Archer.G. E., Friedman, H. S.. Kwalra. M. M., Bigner, S. H., and Bigner, D. D. Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma. Proc. Nat. Acad. Sci. USA, 87:4207-4211, 1990.

8. Garcia de Palazzo, 1., Adams, G. P., Sundareshan, P.. Wong, A. J., Testa, J. R.,Bigner, D. D., and Weiner. L. M. Expression of mutated epidermal growth factorreceptor by non-small cell lung carcinomas. Cancer Res., 53: 3217-3220, 1993.

9. Bigner. S. H., Humphrey, P. A., Wong, A. J., Vogelstein, B., Mark, J., Friedman.H. S., and Bigner. D. B. Characterization of the epidermal growth factor receptor inhuman glioma cell lines and xenografts. Cancer Res., 50: 8017-8022, 1990.

10. Gullick. W. J., Downward. J.. Parker, P. J., Whittle, N., Kris, R., Schlessinger, J.,Ullrich. A., and Waterfield, M. D. The structure and function of the epidermal growthfactor receptor studied by using anti-synthetic peptide antibodies. Proc. R. Soc. Lond.B Biol. Sci., 2256: 127-134, 1985.

11. Wikstrand, C. J., Stanley, S. D., Humphrey, P. A., Pegram, C. N., Archer, G. E.,Kurpad. S., Shibuya, M., and Bigner, D. D. Investigation of a synthetic peptide asimmunogen for a variant epidermal growth factor receptor associated with gliomas. J.Neuroimmunol., 46: 165-174, 1993.

12. Bernatowicz. M. S.. and Matsuada G. R. Preparation of peptide-protein immunogensusing N-succinimidyl bromoacetate as a heterobifunctional cross-linking reagent.Anal. Biochem., 755: 95-102, 1986.

13. Wikstrand, C. J.. Bourdon, M. A., Pegram, C. N., and Bigner, D. D. Human fetal brainantigen expression common to tumors of neuroectodermal tissue origin: gliomas.neuroblastomas, and melanomas. J. Neuroimmunol.. .?: 43-62, 1982.

14. Wikstrand. C. J., McLcndon. R. E.. Bullard. D. E., Fredman, P., Svcnncrholm, L., andBigner, D. D. Production and characterization of two human glioma xenograftlocalizing monoclonal antibodies (MAbs). Cancer Res., 46: 5933-5940, 1986.

15. He, X., Archer, G. E.. Wikstrand, C. J., Morrison, S. L., Zalulsky, M. R.. Bigner.D. D., and Batra. S. K. Generation and characterization of a mouse/human chimcricantibody directed against extracellular matrix protein tenascin. J. Neuroimmunol., 52:127-137, 1994.

16. McPherson, G. A. A practical computer-based approach to the analysis of radioligandexperiments. Comput. Methods Programs Biomed., 17: 107-114, 1983.

17. Lindmo, T., Boven, E., Cuttitta, F., Fedorko, J., and Bunn, P. A. Determination of Iheimmunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J. Immunol. Methods, 72: 77-89, 1984.

18. Bjerkvig, R., Engebraaten, O.. Laerum, O. D.. Fredman. P., Svennerholm, L.. Vrionis,F. D., Wikstrand, C. J., and Bigner, D. D. Anti-GM2 monoclonal antibodies inducenecrosis in GM2-rich cultures of a human glioma cell line. Cancer Res., 51:4643-4648, 1991.

3147

Research. on September 20, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

SPECIFIC raAhs TO EGFRvIII

19. Libcrmann, T. A.. Nusbaum, H. R., Razón,N.. Kris, R.. Lax, I., Soreq. H., Whittle.N., Waterfield. M. D.. Ullrich, A., and Schlessinger. J. Amplificalion, enhancedexpression and possible rearrangement of EGF receptor gene in primary human braintumours of glial origin. Nature (Lond.), 313: 144-147, 1985.

20. Wikstrand, C. J., Friedman, H. S.. and Bigner, D. D. Medulloblastoma cell-substrateinteraction in vitro. Invasion Metastasis, //: 310-324, 1991.

21. Johnsson. B.. Lofas. S., and Lindqvist. G. Immobilization of proteins to a carboxy-methyldcxtran-modified gold surface for biospecific interaction analysis in surfaceplasmon resonance sensors. Anal. Biochem.. I9H: 268-277, 1991.

22. Calakos, N., Bennett. M. K., Peterson, K. E., and Serieller, R. H. Protein-protein

interactions contributing to the specificity of intraccllular vesicular trafficking.Science (Washington DC), 263: 1146-1149. 1994.

23. O'Shannessy. D. J.. Brigham-Burke. M.. Soneson. K. K.. Hensley. P., and Brooks. 1.

Determination of rate and equilibrium binding constants for macromolccular interactions using surface plasmon resonance: use of non-linear least squares analysismethods. Anal. Biochem., 212: 457-468, 1993.

24. Chomczynski, P., and Sacchi, N. Single-step method of RNA isolation by acid••ii.un.inni.MIthiocyanate-phenol-chloroform extraction. Anal. Biochem., 162:156-159, 1987.

25. Pruss. R. M., and Herschman. H. R. Variants of 3T3 cells lacking mitogenic responseto epidermal growth factor. Proc. Nati. Acad. Sci. USA, 14: 3918-3921, 1977.

26. Prabhu, S. C., Batra, S. K., Wikstrand, C. J., Shibuya, M., and Bigner, D. D.Transfection of human epidermal growth factor receptor mutant cDNA into an NIHSwiss 3T3 variant, NR6, cell line: integration, expression, and characterization. Mol.Biol. Cell, 4 (Suppl.).- 21A, 1993.

27. Ullrich, A., Coussens, L., Hayflick, J. S., el al. Human epidermal growth factorreceptor cDNA sequence and aberrant expression of the amplified gene in A431epidermoid carcinoma cells. Nature (Lond.). 309: 418-425, 1984.

28. Batra, S. K., Kern, H. F., Worlock, A. J.. Metzgar, R. S., and Hollingsworth, M. A.Transfection of the human Mud mucin gene into a poorly differentiated humanpancreatic tumor cell line. Panel: integration, expression, and ultrastructural changes.J. Cell Science. 700: 891-894. 1991.

29. Malmborg. A. D.. Michaelsson. A. C., Ohlin, M.. Janssen, B., and Borrebaeck, C. A.Real time analysis of antibody-antigen reaction kinetics. Scand. J. Immunol., 35:643-650, 1992.

30. Karlsson, R., Michaelsson. A., and Mattsson, L. Kinetic analysis of monoclonalantibody-antigen interactions with a new biosensor based analytical system.J. Immunol. Methods, 145: 229-240, 1991.

31. Reeves, J. R., Cooke, T. G., Fenton-Lee, D., McNicol. A. M.. Ozanne, B. W.,

Richards, R. C., and Walsh. A. Localization of EGF receptors in frozen tissue sectionsby antibody and biotinylatcd EGF-based techniques. J. Histochem. Cytochem., 42:307-314. 1994.

32. Marti, U., and Gebhardt, R. Acinar heterogeneity of the epidermal growth factorreceptor in the liver of male rats. Eur. J. Cell Biol., 55: 158-164. 1991.

33. Foresta, C., Carello, A., Varotto, A., Rossato, M., and Scandellari. C. Epidermalgrowth factor receptors (EGFR) localization in human lestis. Arch. Androl., 27:17-24, 1991.

34. Owens. O. J., and Leake, R. E. Epidermal growth factor receptor in normal ovariesand benign ovarian tumors. Eur. J. Obstet. Gynecol. Reprod. Biol., 47: 229—233,

1992.35. Zeder-Lutz, G., Altschuh, D., Geysen, H. M.. Trifilieff, E., Sommermeyer, G., and

van Rcgenmortel, M. H. V. Monoclonal antipeptide antibodies: affinity and kineticrate constants measured for the peptide and the cognate protein using a biosensortechnology. Mol. Immunol., 30: 145-155, 1993.

36. Kaufman, E. N.. and Jain, R. K. Effect of bivalent interaction upon apparent antibodyaffinity: experimental confirmation of theory using fluorescence photobleaching andimplications for antibody binding assays. Cancer Res.. 52: 4157-4167, 1992.

37. Fujimori, K., Covell, D. C., Fletcher, J. E., and Weinstein, J. N. A modeling analysisof the global and microscopic distribution of IgG. F(ab')2, and Fab in tumors. Cancer

Res., 49: 5656-5663, 1989.38. Fujimori, K., Fisher. D. R., and Weinstein. J. N. Integrated microscopic-macroscopic

pharmacology of momx'lonal antibody radioconjugates: the radiation dose distribution. Cancer Res., 51: 4821-4827, 1991.

39. Van Osdol, W.. Fujimori, K.. and Weinstein, J. N. An analysis of monoclonalantibody distribution in microscopic tumor nodules: consequences of a "binding sitebarrier." Cancer Res., 5/: 4776-4784, 1991.

40. Langmuir, V. K., Mendonca. H. L., and Woo. D. V. Comparisons between twomonoclonal antibodies that bind to the same antigen but have differing affinities:uptake kinetics and '25I-antibody therapy efficacy in multiceli spheroids. Cancer Res.,

52: 4278-4784, 1992.41. Nishikawa, R., Ji, X-D., Harmon, R. C., Lazar, C. S., Gill, G. N., Cavenee, W. K.,

and Huang, H-J. S. A mutant epidermal growth factor receptor common in

human glioma confers enhanced tumorigenicity. Proc. Nati. Acad. Sci. USA,9/.-.7727-7731, 1994.

42. Lorimer. I. A. J.. Wikstrand, C. J., Batra, S. K.. Bigner, D. D., and Pastan, I.Immunotoxins that target an oncogenic mutant epidermal growth factor receptorexpressed in human tumors. Clin. Cancer Res., in press, 1995.

43. Modjtahedi. H., Eccles, S.. Sandle. J., Box, G.. Titley, J., and Dean, C. Differentiationor immune destruction: two pathways for therapy of squamous cell carcinomas withantibodies to the epidermal growth factor receptor. Cancer Res., 54: 1695-1701,

1994.44. Vitella, E. S., and Uhr. J. W. Monoclonal antibodies as agonists: an expanded role for

Iheir use in cancer therapy. Cancer Res., 54: 5301-5309, 1994.

3148

Research. on September 20, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

1995;55:3140-3148. Cancer Res   Carol J. Wikstrand, Laura P. Hale, Surinder K. Batra, et al.   Gliomasand React with Breast and Lung Carcinomas and Malignant Monoclonal Antibodies against EGFRvIII Are Tumor Specific

  Updated version

  http://cancerres.aacrjournals.org/content/55/14/3140

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/55/14/3140To request permission to re-use all or part of this article, use this link

Research. on September 20, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from