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HYBRIDOMA AND HYBRIDOMICS Volume 21, Number 3, 2002 © Mary Ann Liebert, Inc.

Generation and Characterization of Polyclonal Antibodies Specific for Human p110 sEGFR TRACE A. CHRISTENSEN, JILL L. REITER, ANDRE T. BARON, and NITA J. MAIHLE

ABSTRACT The EGFR/ERBB family of receptor tyrosine kinases mediates intracellular signal transduction pathways important in the regulation of cell growth, differentiation, and transformation. We previously have reported the cloning and expression of a 3 kb alternative EGFR transcript which encodes a 110 kDa form of the receptor (p110 sEGFR). This receptor isoform is identical to the extracellular region of the full-length 170 kDa EGFR through amino acid 603; in addition, p110 sEGFR contains 78 unique carboxy-terminal amino acids. Here, we report the generation and characterization of polyclonal antisera specific for the unique carboxy-terminal sequence of p110 sEGFR. Polyclonal antisera were generated by immunizing rabbits with synthetic peptides corresponding to peptides contained within the unique carboxy-terminal sequence of p110 sEGFR. Immunoglobulin fractions from antisera which tested positive for immune reactivity to these peptides by ELISA were affinity-purified by protein G and peptide-based chromatography. This affinity-purified immunoglobulin fraction specifically recognizes p110 sEGFR by ELISA, immunoprecipitation, immunoblot analysis, and immunocytochemical methods. No cross-reactivity with full-length p170 EGFR is observed using any of these detection methods. These new polyclonal antibodies will be useful in determining the expression, localization, and function of p110 sEGFR, and importantly will allow us to distinguish between the expression of this receptor isoform and p170 EGFR. INTRODUCTION

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GFR IS THE PROTOTYPIC MEMBER of the EGFR/ERBB family of receptor tyrosine kinases, which includes EGFR (ERBB1/HER1), ERBB2 (HER2/NEU), ERBB3 (HER3), and ERBB4 (HER4). These receptors play an important role in the control of diverse biological processes including cell survival, proliferation, differentiation, adhesion, migration, and malignant transformation. Overexpression of EGFR in human carcinomas is frequently associated with resistance to radiation and chemotherapy, disease recurrence, and hence, overall poor patient prognosis.(1–3) Additionally, tumors frequently overexpress EGF family growth factors, leading to autocrine stimulation of mitogenesis and to a loss of growth control. Consequently, EGFR and its isoforms are being investigated as tumor biomarkers and also as potential targets for the development of novel anticancer therapies.(3–5) EGFR structure is characterized by an extracellular ligandbinding domain, a transmembrane-spanning domain, an intracellular domain with intrinsic tyrosine kinase activity, and a carboxy-terminal regulatory region. In addition to the tran-

scripts (5.8 and 10 kb) encoding the full-length 170 kDa EGFR, several naturally occurring alternative transcripts of the human EGFR gene have been reported that encode for truncated proteins containing only the ligand-binding domain of this receptor.(6,7) In this study, we focus on one such truncated EGFR isoform, which we have designated p110 sEGFR, that is encoded by the 3 kb alternatively spliced human EGFR transcript.(7) This 110 kDa isoform shares complete sequence homology with the full-length 170 kDa EGFR until amino acid 603, at which point the two sequences diverge; the 110 kDa sEGFR isoform contains a unique 78 carboxyl-terminal amino acid sequence that is not present in the 170 kDa EGFR sequence. Here, we report the generation and characterization of rabbit polyclonal antisera specific for p110 sEGFR. These antisera were prepared by immunizing rabbits with peptides corresponding to the unique 78 amino acid region of p110 sEGFR. The specificity of these antisera was established by competition binding studies using enzyme-linked immunosorbant assay (ELISA), immunoprecipitation, immunoblot, and immunohistochemical analysis. This antibody should prove to be useful

Tumor Biology Program, Mayo Clinic, Rochester, MN 55905.

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for distinguishing between the expression and localization of p110 sEGFR and full-length p170 EGFR in future studies.

MATERIALS AND METHODS Peptide synthesis and conjugation Peptides A (amino acids #604-634) and B (amino acids #620634) were synthesized by the Mayo Clinic Protein Core Facility using an automated peptide synthesizer (Advanced ChemTech Inc., Louisville, KY) according to the manufacturer’s instructions. Peptides were purified by reverse-phase HPLC and analyzed by mass spectrometry to confirm their identity. Peptide A was conjugated to keyhole limpet hemocyanin (KLH, Sigma Chemical Co., St. Louis, MO) and peptide B was synthesized using an 8-branch lysine core MAPS residue (Calbiochem-Novabiochem Corp., San Diego, CA). The numbering system for peptides A and B corresponds to the amino acid sequence of p110 sEGFR.(7)

Antisera production Antisera was generated by Cocalico Biologicals, Inc. (Reamstown, PA) in female New Zealand White (NZW) rabbits. Prior to immunization, serum samples were screened to verify that no pre-existing antibodies toward p110 sEGFR were detectable by ELISA and immunoblot methods. Two rabbits each were immunized with either 100 mg of KLH-conjugated peptide A or 100 mg of MAPS-conjugated peptide B. Rabbits were boosted with 50 mg of conjugated peptides at 2, 3 and 7 weeks post inoculation with additional boosts every 4 weeks thereafter. Serum samples were collected from rabbits two weeks after each boost injection and monitored for an immune response to each peptide by ELISA.

Peptide-based affinity chromatography Peptide affinity columns for both peptides A and B were prepared similarly using Sulfolink coupling gel (Pierce, Rockford,

IL). Briefly, unconjugated peptide containing a terminal cysteine residue was irreversibly immobilized to the agarose supporting gel via free sulfhydryl groups according to the manufacturer’s recommendations. Antiserum was cleared of lipids using SeroClear Reagent or PHM-L Liposorb (CalbiochemNovabiochem Corp.) according to manufacturer’s instructions. Total serum immunoglobulins were isolated using HiTrap affinity protein G columns (Amersham Pharmacia Inc., Piscataway, NJ) according to the manufacturer’s instructions and concentrated to approximately 1 mL by ultrafiltration with Centriprep30 and Centricon-30 centrifugal concentrators (Millipore Corp., Bedford, MA). The concentrated immunoglobulin fraction was bound to the peptide affinity column in 20 mM sodium phosphate, pH 7.0, eluted with 0.1 mM glycine-HCl, pH 2.7, and neutralized with 1 M Tris-HCl, pH 9.0. Eluted immunoglobulins were concentrated and exchanged into 10 mM potassium phosphate, pH 7.4, 150 mM NaCl, 0.04% NaN3 and analyzed for their ability to bind cognate peptide by ELISA. Enzyme-linked immunosorbant assay. Rabbit antisera and purified immunoglobulin fractions were tested for responsiveness to peptides by a modification of an ELISA method described previously.(8) Pro-bind 96-well microtiter plates (Falcon/BD Biosciences, Franklin Lakes, NJ) were coated with 10 mg/mL of unconjugated peptide A or B and incubated at room temperature overnight. Plates were washed with Tris buffered saline (TBS; 10 mM Tris, pH 7.4, 150 mM NaCl), blocked with ELISA blocking buffer (ELBB; 0.1% BSA and 0.02% NaN3 in TBS), rinsed briefly with TBS, incubated with serial dilutions of antisera or affinity-purified Ig, washed again with TBS, incubated with alkaline phosphatase-conjugated goat anti-rabbit Ig antibody (ICN Biomedicals Inc., Aurora, OH), washed with TBS, and reacted with p-nitrophenyl phosphate. For peptide competition experiments a dilution series of peptide A was preincubated with a constant concentration of antibody 1 h prior to analysis. All assays were performed at room temperature with a BIOMEK 1000 automated laboratory workstation (Beckman Instruments, Inc., Palo Alto, CA) equipped with a photometry

FIG. 1. A diagrammatic illustration of full-length p170 EGFR and p110 sEGFR. The unique 78 amino acid carboxyl-terminal sequence of p110 sEGFR is shown, with bars indicating the specific amino acid sequences (peptide A #604-634 or peptide B #620-634) used to generate polyclonal antisera described in this report.

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tool. Optical density readings were recorded at 15 min intervals for 1.5 h at an absorbance of 405 nm.

a high and uniform level of p110 sEGFR expression and was expanded for further study.

Transfected cell lines

Metabolic labeling and immunoprecipitation

Chinese hamster ovary (CHO) cells (which express no endogenous EGFR) were transfected with the expression vector pcDNA3 (Invitrogen, Carlsbad, CA) (CHO/vector), or with the vector containing cDNA encoding the full-length p170 EGFR (CHO/p170), or the 3 kb EGFR cDNA (pcDNA2241) encoding p110 sEGFR (CHO/p110).(7) Stable CHO/p110 cell lines were selected for G418 resistance and clonal isolates were screened for p110 sEGFR expression by immunoblot analysis and immunofluorescence microscopy, using antibodies specific for the extracellular domain of the EGFR. Clone 16.9 showed

Approximately 8 3 106 cells were metabolically labeled in methionine-free DMEM supplemented with 80 mCi/mL [35S]methionine (EasyTag, Dupont/NEN, Wilmington, DE) for 4 hrs at 37°C in 5% CO2 and air. Cell monolayers were washed with cold phosphate-buffered saline (PBS; 10 mM KH2PO/Na2HPO4, 0.1 M NaCl, 2.7 mM KCl, pH 7.4) lysed in SDS lysis buffer, and immunoprecipitated with mAb 15E11(8) or polyclonal anti-p110 sera as described previously.(9) For peptide competition experiments, affinity-purified anti-p110 Ig was preincubated with 2 mg/mL peptide A for 30 min at room tem-

FIG. 2. ELISA characterization of affinity-purified p110 sEGFR antisera. Dilution series of affinity-purified anti-peptide A Ig, total anti-peptide A Ig, affinity column wash fractions, and total pre-immune Ig reacted against cognate peptide (A). Similar fractions were prepared from antiserum of peptide B inoculated rabbits purified with peptide B affinity column (B). Competition ELISA using a dilution series of peptide A starting at 100 mg/mL preincubated with constant concentration (2 mg/mL) of affinity-purified antipeptide A Ig (C).

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Immunocytochemistry Cells were plated onto glass coverslips and allowed to grow under standard culture conditions for 24 h. Cells were washed with PBS supplemented with calcium and magnesium (1 mM CaCl2 and 0.5 mM MgCl2) and fixed in methanol for 10 min at 220°C. Following fixation, cells were air-dried and rehydrated in PBS. Cells were blocked using immunofluorescence blocking buffer (IFBB, PBS with 5% goat serum, 1% glycerol, 0.1% BSA, 0.1% gelatin) for 10 min and incubated for 1 h at room temperature with 3 mg/mL affinity purified anti-p110 sEGFR Ig or total pre-immune Ig. Following a 5 min wash in PBS, cells were incubated with a biotinylated goat anti-rabbit secondary antibody (Vector Labs Inc., Burlingame, CA) diluted 1:2000 in IFBB for 20 min at room temperature. Labeling was detected using the Vectastain Elite ABC Kit (Vector Labs Inc.), according to manufacturer’s instructions. Cells were counterstained with Mayer’s hematoxylin and mounted using Glycergel mounting medium (Vector Labs Inc.). Micrographs were obtained using a Zeiss Axioplan2 microscope equipped with an AxioCam digital camera and Zeiss KS400 image acquisition software (Zeiss, Thornwood, NY).

RESULTS Peptide selection for immunization

FIG. 3. Immunoprecipitation of p170 EGFR and p110 sEGFR isoforms. Transfected CHO cell lines were metabolically labeled with [35S]methionine, and cell lysates were immunoprecipitated with either affinity-purified anti-p110 sEGFR (A), or MAb 15E11 (B). Lane 1, (CHO/vector); lane 2, (CHO/p170); lane 3, (CHO/p110); lane 4, CHO/p110 competed with peptide A.

perature prior to immunoprecipitation. Immunoprecipitated proteins were resolved by SDS-PAGE.(10) Gels were stained with Coomassie brilliant blue in 50% methanol and 7% acetic acid, destained with 50% methanol and 7% acetic acid, treated with Amplify (Amersham Pharmacia Inc.), vacuum dried and processed for fluorography with film (Kodak, Rochester, NY) at 270°C.

Immunoblot analyses Cell lysates were prepared, resolved by 7.5% SDS-PAGE and immunoblotted as described previously.(8,9,11) A 1:500 dilution of affinity purified anti-p110 sEGFR Ig or a 1:10 dilution of conditioned culture medium from 15E11 hybridoma cells(8) were used as primary antibodies. A 1:20,000 dilution of HRP-conjugated goat anti-rabbit or goat anti-mouse antibodies (ICN Biochemicals, Aurora, OH) were used as secondary antibodies. Antibody binding was visualized and recorded using the Supersignal West Femto chemiluminescent detection substrate (Pierce Inc.) and a Nucleovision gel documentation system (NucleoTech Corp., Hayward, CA).

To generate antisera which can distinguish between p110 sEGFR and the full-length p170 EGFR, we selected two peptides from within the unique 78 amino acid carboxy-terminal sequence of p110 sEGFR to serve as antigens for immunizing rabbits (Fig. 1). Peptide A (amino acids #604-634) corresponds to the first 31 amino acids of this unique sequence, while peptide B (amino acids #620-634) spans the carboxy-terminal 15 amino acids of peptide A (Fig. 1).

ELISA analyses Rabbits immunized with either peptides A or B were monitored for immune response by ELISA. Serum samples from rabbits immunized with peptide A or B yielded positive dose response curves against their cognate peptide following the second boost injection with antigen (data not shown). Total immunoglobulin fractions from both the pre-immune and immune sera of each rabbit were isolated by protein G chromatography. Immunoglobulins specific for either peptides A or B were isolated further from the total Ig fractions using peptide affinity columns. Figure 2 shows ELISA dose response curves of particular anti-peptide Ig column fractions toward cognate peptide. Positive dose responses against cognate peptide were observed with total immune Ig and affinity-purified Ig specific for peptide A (Fig. 2A) or peptide B (Fig. 2B). No specific binding was observed in either total pre-immune Ig or affinity column wash fractions. To demonstrate further the specificity of the anti-p110 sEGFR Ig purified from the peptide A affinity column, we performed competitive binding ELISA experiments using peptide A as the competitive inhibitor. Competitive binding of anti-peptide-A affinity-purified Ig produced a diminished dose response (Fig. 2C). All subsequent antisera characterization in this re-

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FIG. 4. Immunoblot analysis of total cell proteins from CHO/vector, CHO/p170, and CHO/p110. Duplicate blots were incubated with either anti-p110 sEGFR (A), or MAb 15E11 (B). Lane 1, CHO/vector; Lane 2, CHO/p170; Lane 3, CHO/p110.

port refers to the affinity-purified fraction from the peptide A affinity column and is designated as anti-p110 sEGFR.

Immunoprecipitation To test the ability of anti-p110 sEGFR to discriminate between p110 sEGFR and p170 EGFR, CHO cells transfected with either p110 sEGFR or p170 EGFR were metabolically labeled with [35S]methionine and cell lysates were immunoprecipitated with anti-p110 sEGFR. This affinity-purified antibody specifically immunoprecipitated p110 sEGFR from the CHO/p110 cell line but did not show any cross reactivity with p170 EGFR (Fig. 3A). In contrast, MAb 15E11, which specifically recognizes the extracellular domain of EGFR,(8) immunoprecipitated both p170 EGFR and p110 sEGFR from CHO/p170 and CHO/p110 cell lines, respectively (Fig. 3B). To demonstrate further the specificity of the anti-p110 sEGFR we performed a competitive binding experiment using

peptide A as the competitive inhibitor. Preincubation of antip110 sEGFR with unconjugated peptide A significantly decreased the ability of this antibody to immunoprecipitate p110 sEGFR from CHO/p110 whole cell lysates (Fig. 3A, lane 4). In contrast, peptide A did not reduce the ability of MAb 15E11 to immunoprecipitate p110 sEGFR from CHO/p110 whole cell lysates (Fig. 3B, lane 4). These data indicate that anti-p110 sEGFR specifically immunoprecipitates p110 sEGFR, and does not cross react with p170 EGFR by this method of analysis.

Immunoblot analyses The ability of anti-p110 sEGFR to recognize specifically p110 sEGFR in transfected CHO cells was further assessed by immunoblot analysis. Total protein from CHO/vector, CHO/p170, and CHO/p110 cells was resolved by SDS-PAGE, transferred to PVDF membrane, and reacted with either antip110 sEGFR (Fig. 4A) or mAb 15E11 (Fig. 4B). Anti-p110

FIG. 5. Immunocytochemical analysis of transfected CHO cells using anti-p110 sEGFR. CHO/vector (A), CHO/p170 (B), CHO/p110 (C) were processed using avidin/biotin peroxidase-based immunocytochemistry with AEC as the visualizing chromagen. Micrographs were taken at 10003 magnification.

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sEGFR recognized p110 sEGFR, while 15E11 recognized both p110 sEGFR and p170 EGFR, further documenting the specificity of this polyclonal antisera.

Immunocytochemical analyses To determine the utility of anti-p110 sEGFR for immunocytochemistry, we labeled methanol fixed CHO/vector, CHO/p170, and CHO/p110 cells using an avidin/biotin peroxidase-based protocol (Fig. 5). Only CHO/p110 cells reacted with anti-p110 sEGFR (Fig. 5C). Under these labeling conditions, p110 sEGFR is predominantly localized in the cytoplasm, where it apparently is associated with the endoplasmic reticulum. Similar experiments with p170 EGFR in the CHO/p170 cells (Fig. 5B) revealed no significant cross-reactivity, or background staining in the CHO/vector cells which had been processed identically (Fig. 5A).

tibody that can specifically distinguish between this sEGFR isoform and the full-length EGFR should provide a valuable reagent for future studies on expression and function of this novel receptor isoform in diverse human tissues.

ACKNOWLEDGMENTS We gratefully acknowledge Dan McCormick and the Mayo Protein Core Facility for assisting in the selection and generation of immunizing peptides. We thank Jackie Lafky for assisting with the column chromatography and Steve Zeismer for technical help with performing the immunocytochemistry. This work was supported by NIH grants CA76170 and CA85133, and by a Regis Foundation Breast Cancer Research Scholarship.

REFERENCES DISCUSSION Recent studies of soluble growth factor receptors have highlighted the significant role of these receptors in the regulation of processes essential for cell growth and survival, either in conjunction with, or independent of, expression of their related fulllength receptors.(12) The EGFR/ERBB family of receptor tyrosine kinases is one of several growth factor receptor families which express soluble receptor isoforms. Our laboratory has cloned several naturally occurring alternative transcripts of the human EGFR gene, and we have begun to characterize the function, localization and expression of these receptor isoforms.(6,7) Although the functional importance of these soluble EGFR isoforms has not yet been fully elucidated, in vitro analysis has shown that sEGFR receptor isoforms can function to block ligand-dependent signaling, including oncogenic transformation. (13) These are important observations, given that EGFR isoforms are expressed in a wide spectrum of human tissues and can be detected at measurable levels in the sera of healthy individuals, as well as in the sera of cancer patients.(11,14,15) Presently, expression and localization studies of soluble EGFR isoforms are limited to existing antibodies to the extracellular domain of EGFR. Structural similarities between EGFR isoforms make it difficult to distinguish full-length p170 EGFR from any of the soluble isoforms using these antibodies. In this report, we have taken advantage of the unique structural characteristics of p110 sEGFR to develop antibodies which specifically recognize this isoform. We report here the successful generation and characterization of polyclonal antisera specific for p110 sEGFR. Immunoprecipitation, immunoblot, and immunocytochemical analyses demonstrate that an anti-p110 sEGFR affinity purified Ig preparation specifically recognizes p110 sEGFR, and importantly, does not cross-react with full-length p170 EGFR or with any of the other sEGFR isoforms that we have identified.(7) Studies are currently underway to further explore the functional relationship between p110 sEGFR and its full-length receptor counterpart, p170 EGFR. Given the unique structural characteristics of p110 sEGFR, its tissue-specific expression in human tissues,(7) and the well established role of EGF family ligands in signaling diverse biological endpoints, the importance of generating an an-

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14. Baron AT, Lafky JM, Boardman CH, Balasubramaniam S, Suman VJ, Podratz KC, and Maihle NJ: Serum sErbB1 and epidermal growth factor levels as tumor biomarkers in women with stage III or IV epithelial ovarian cancer. Cancer Epidemiol Biomarkers Prev 1999;8:129–137. 15. Baron AT, Lafky JM, Suman VJ, Hillman DW, Buenafe MC, Boardman CH, Podratz KC, Perez EA, and Maihle NJ: A preliminary study of serum concentrations of soluble epidermal growth factor receptor (sErbB1), gonadotropins, and steroid hormones in healthy men and women. Cancer Epidemiol Biomarkers Prev 2001;10:1175 –1185.

Address reprint requests to: Nita J. Maihle, Ph.D. Tumor Biology Program Mayo Clinic 200 First Street SW Rochester, MN 55905 E-mail: [email protected] Received for publication January 24, 2002. Accepted for publication February 25, 2002.