HYBRIDOMA Volume 11, Number 6, 1992



Liebert, Inc., Publishers

Production of Anti-Fibroblast Growth Factor Receptor Monoclonal Antibodies by In Vitro Immunization SUBRAMANIAM VENKATESWARAN, VINCENT and MARGARET SCHELLING


Department of Genetics and Cell Biology, Wishington State University, Pullman,

Wi 99164-4234


Monoclonal antibodies against the high affinity tyrosine kinase Fibroblast Growth Factor (FGF) receptor may help define receptor epitopes ¡nuolued in FGF binding and signal transduction which mediate coronary and tumor angiogenesis (the deuelopment of blood uessels). Monoclonal antibodies against the FGF receptor mere made by in uitro immunization. Receptor mas PRGE-purified and transblotted to nitrocellulose. The nitrocellulose was dissolued with acetonitrile, and the receptor used as antigen in an in uitro immunization system. Screening for FGF receptor positiue clones was done using membrane preparations from Coronary Uenular Endothelial Cells (CUEC) expressing the FGF receptor, both by Enzyme Linked Immunosorbent Rssay (ELISR) and Western blot. Culture supernatants of seueral clones tested positiue for IgM or IgG monoclonal antibodies against the FGF receptor. Rntibodies were affinity purified. Rscites fluid was produced in Balb/c mice primed with Incomplete Freund's adjuuant (IFR). The monoclonal antibodies were also found to be suitable for receptor immunoprecipitation. Immunocytochemistry was done on sections from a uariety of species. Further characterization of these antibodies, as well as the production of the remainder of a panel of anti-FGF receptor monoclonal antibodies, is underway. INTRODUCTION The application of monoclonal antibody technology to examining cell surfaces has yielded new strategies for the isolation and characterization of cell membrane receptors. Monoclonal antibodies are uniquely useful as probes in both functional and structural studies of receptors. Many systems (1-8) haue documented the usefulness of monoclonal antibodies in quantitating membrane receptors and studying receptor function in a wide uariety of cell types. Monoclonal antibodies are useful for receptor isolation by immunoaffinity chromatography and immunoprecipitation. Additionally, anti-receptor monoclonal antibodies can be used to identify the products of translation of receptor-specific mRNfl, for receptor turn-ouer studies, and for immunopreciptiation followed by direct sequencing of intrinsically radioactiuely labelled receptor. Obtaining sufficient purified receptor for the production of monoclonal antibodies in response to growth factor receptors is frequently an obstacle to receptor research. Ole haue circumuented this problem by the utilization of in uitro immunization in combination with PRGE-purified receptor to produce 729

monoclonal antibodies against the Fibroblast Growth Factor receptor. In uitro immunization inuolues the isolation of spleen cells from a donor mouse followed by the "in uitro" or "in tissue culture" addition of antigen to the spleen cells. "Immunization" therefore occurs in the tissue culture flask rather than in the mouse. Much less antigen is required with in uitro immunization, and it is possible

antibodies in response to highly conserued molecules. In uitro an excellent mechanism for the production of monoclonal antibodies to highly conserued molecules unauailable in large quantities such as growth factors and their receptors.



immunization prouides

MRTERIRLS RND METHODS Culture of Coronary Uenular Endothelial Cells (CUEC) beads as CUECs were isolated from postcapillary uenules utilizing 15 µ preuiously described (9). Endothelial cells were maintained in complete DMEM (CDMEM) [Dulbecco's Modified Eagle's Medium (M.R. Bioproducts) + 2mM sodium pyruuate, 2mM L-glutamine (Hazelton, Denuer, PR), 100 lU/ml penicillin, 100 ug/ml

streptomycin (M.R. Bioproducts, IDalkersuille, MD) and 2.5 /ig/ml Fungizone (Flow, McLean, UR)1 supplemented with 20 % fetal calf serum and 2.6 /ig/ml heparin (Sigma). Gelatin (Difco, Detroit, Ml) was used as the tissue culture matrix. In

addition to exhibiting typical endothelial cobblestone morphology, the CUECs were confirmed to be endothelial by staining positiue for factor Ulli antigen and UER lectin (9). CUEC membrane fragments containing high affinity FGF receptors (10-12) were prepared according to our published protocol (13). Production of Monoclonal Rntibodies The CUEC membrane preparation FGF receptor was crosslinked to i25l-FGF with the reuersible crosslinker Ethylene glycol Bis(succinimidylsuccinate) (EGS) on SDS-PRGE to identify the receptor molecule (11). The receptor was then uncoupled with hydroxylamine for 3-6 hrs at 37°C and transblotted to nitrocellulose paper (NCP). The receptor protein was recouered from the NCP using 50% acetonitrile in methanol (14). The PRGE-purified receptor was then dissolued in a minimum uolume of sterile PBS containing complete Freund's adjuuant (1:1) and injected I.P. into Balb/c mice. Subsequent injections were giuen at 2 week interuals by the same route of injection using incomplete Freund's adjuuant. Three days after the booster dose, the spleen cells were used for in uitro immunization (15) (donor spleen cells were combined with thymocyte conditioned medium and purified receptor (day 0) according to our preuiously published method) and subsequent fusion (day 3-5) with myeloma cells according to our preuiously published =

protocol (16).

The BRLB/c nonsecreting myeloma line SP2/0-RG14 was obtained from the Rmerican Type Culture Collection (CRL-1581). Myeloma and hybridoma cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (M.R. Bioproducts) supplemented (SDMEM) with 4.5 g/l glucose, 2mM L-glutamine, 2mM sodium pyruuate, 2.5 /ig/ml fungizone (Flow), IBB /ig/ml garamycin (Sigma, St. Louis, M0) For the preparation of thymocyteand 20% fetal bouine serum (Hyclone). conditioned medium (TCM), thymocytes from BRLB/c mice were cultured at 5 IB6 cells/ml in DMEM supplemented with 2 mM sodium pyruuate, 2 mM L-glutamine, 10 /iM MeM nonessential amino acids (M. R. Bioproducts) and 2% fetal bouine serum (CDMEM). Rfter 48 h of incubation at 37°C, 7% CO2, cells were remoued by

centrifugatlon (15 min., Screening of



and the TCM was stored at


Clones for Rnti-FGF Receptor Monoclonal Rntibodies:


Linked Immunosorbent Rssay Nunc immunoplates were coated with the membrane preparation (13) at a protein concentration of 2 /ig/50 /il/well in Tris buffered saline (TBS), pH 7.4. The plates were left couered at 4°C in a cold room until they were dry and were stored at -20°C until used in assays. The plates to be used for the assay were allowed to


to room temperature. The plates were initially blocked with ßµ /well of 2% BSR in Tris buffered saline (20 mM Tris containing 200 mM NaCI, pH 7.4) at room temperature for 4 hr. followed by ouernight at 4°C. The blocking solution was tapped off and the plates incubated with the appropriate culture supernatants for testing, undiluted, at room temperature for 4 hr. or ouernight at 4°C. The plates were then washed in TBS containing 0.1% BSR, 3-5 times and incubated with appropriate biotinylated secondary antibody, (1:750, Rmersham), 50 /il/well for 90 min. at room temperature, washed as aboue and incubated with streptauidinbiotin peroxidase conjugate (1:750, Rmersham), 50 µ /well for 90 min. at room temperature. The excess conjugate was washed off as aboue. The plates were then deueloped using 50 µ /well of ortho-phenylenediamine (0PD) tablets (9 mg/25 ml) dissolued in 58 mM sodium citrate buffer, pH 4.5 containing 16 µ of 30% H2O2- The enzyme reaction was arrested with 0.1 M HCI (50 µ /well) and the color deueloped was measured using an ELISR reader at 490 nm. Rabbit anti FGF receptor (1:150) serued as the internal positiue control. come

western blot analysis western blot analyses



as detailed elsewhere


Hscites Production IO6 cells/ml) The clones found positiue by ELISR were each injected (~1-3 into mice preuiously primed with Incomplete Freund's adjuuant (at least a week earlier as per the protocol) (17). The ascites, produced 2-3 weeks later, was kept at 4°C ouernight, centrifuged, aliquoted, and stored at -70°C. Characterization of the Monoclonal Rntibodies: Purification of the Monoclonal Rntibodies The culture supernatant containing the antibody was dialysed against PBS ouernight at 4°C to remoue serum components and passed through an affinity column containing Con R Sepharose-4B (LKB Pharmacia) preuiously equilibrated with at least ten column uolumes of PBS buffer, pH 7.4, to remoue all the preseruatiue that comes with the column resin. The speed of the column was adjusted to 24 ml/hr. IgM antibody bound to the affinity column was then eluted with 0.2M alpha-methylmannoside (18). 2ml fractions were collected and protein concentration in each tube was measured at 280 nm. The protein from the bound fraction was then tested for the presence of IgM antibodies using the Rmersham Immunotype kit as per the manufacturer's protocol (Fig. 2).

Immunoprecipitation The CUECs were incubated with 50 ng/mL of bFGF for 1 hr. prior to membrane preparation as preuiously described (13). The membranes were solubilized with TBS-2% Triton X100 and the insoluble material was centrifuged at 25,000g, 1 hr., 4°C. The membranes (20B /ig) were incubated with 24 ßg/m\ of rabbit antiphosphotyrosine antibody ouernight at 4°C in TBS pH 7.5 containing 1% Triton K1B0 (final concentration). The protein R-Sepharose was washed in TBS containing IM NaCI and rinsed in 50 mM phosphate buffer pH 7.5 three times before adding a 50 mM phosphate buffer pH 7.5 containing 190 mM sodium chloride. Rt the end of the incubation of the rabbit anti-phosphotyrosine antibody with the CUEC membranes, the protein R-Sepharose was added so that there was a 2x excess of protein RSepharose in relation to the antibody concentration. The protein R-Sepharose and the membrane protein-antibody complex were incubated for 4 hr. at 4°C, centrifuged at the end of the incubation at 1000 g for 20 min. at 4°C, and the supernatant remoued. The pellet was washed 3x with 50 mM phosphate buffer containing 0.5 M NaCI, 1% Triton «100 and twice with 5B mM phosphate buffer containing 190 mM NaCI. The final pellet was then monitored by gel electrophoresis: 33 µ of tris buffer and 17 µ of sample buffer containing 2% Bmercaptoethanol were added, samples were boiled at 100°C for 3 min. and loaded onto a 10% polyacrylamide gel (19). After electrophoresis the proteins were electrotransferred and dyed with amido-black. The membrane was blocked for non 731

specific binding with 5% BSR. The immunoprecipitates obtained with rabbit antiphosphotyrosine antibody were then tested with different antibodies: a) mouse anti-FGF receptor (monoclonal clone UBSI) (culture supernatant), and b) antimouse anti-phosphotyrosine (1:1000). The incubation time was 4 hr. at room temperature for these two antibodies. The second antibodies were incubated with the preparation ouernight at 4°C and were respectiuely: c) Goat anti-mouse IgM biotinylated for UBSI (1:1000) and d) Goat anti-mouse IgG biotinylated, for antiphosphotyrosine (1:1000). The streptauidin-peroxidase complex (1:750) was then added, incubated for 2 hr., and the deueloping was done as preuiously described (13). Molecular Weight Determination of the Rntibody To ßßµ culture supernatant, IBB µ of goat anti mouse IgM solution was added, shaken well for 2 hr. in the cold room, centrifuged at 2000 rpm for 5 min., supernatant aspirated, button of the complex washed twice with PBS by carefully resuspending and centrifuging. Similar precipitates were prepared using normal mouse serum/goat anti mouse IgM and a control antibody. To the precipitate, 2550 µ sample buffer was added and followed by the usual protocol for SDS-PRGE (19). The gels were then siluer stained according to manufacturer's protocol



of the Monoclonal


mouse immunotype kit as


monoclonal antibody was Isotyped per the manufacturer's protocol.

Isoelectrofocusing (IEF) IEF


performed (20)

on a

Mini Protean Unit





the Monoclonal Rntibody Frozen sections of tissues, and CUEC cells, both fixed in -2B°C acetone, used to test the antibody staining pattern as per the published protocol (21).

Immunocytochemical Staining Using


Radioiodination of bFGF 3 /ig of highly purified bFGF purchased from R&D System was radioiodinated using the iodogen technique (Pierce) as per the manufacturer's protocol. Basic FGF was then loaded onto a heparin Sepharose column and eluted with 2M NaCI. The non-retained fraction on heparin was fractionated with an Excellulose™ GF-5 column. The specific actiuity (/iCi//ig) was calculated by diuiding the radioactiuity of the protein by the total quantity of protein.

Binding Rssay

CUEC cells were grown until confluent in 24 well plates, washed IK with cold PBS and IK with ice cold binding buffer (2B mM HEPES pH 8.2, ImM MgCI2, 1 mM CaCl2> 1/ig/ml PMSF, 1 /ig/ml aprotinin and 0.1% BSR) for 30 min. before competitiue binding studies. The competitiue binding studies were carried out as described by Schreiber et al. (22). 10 pM of ' 251-bFGF (SA:~25/iCi//ig) was incubated with or without increasing amounts of unlabeled proteins ranging from 10-·' to 23 nM. The unlabeled proteins were bFGF (homologous displacement), UBSI monoclonal antibody, and a control mouse IgM (heterologous displacement). The cells were incubated for 4 hr at 4° C. The medium was discarded and cells were washed 1 with binding buffer. The cells were disrupted with 0.3 M NaOH in H2O and the cell associated radioactiuity was counted on a Gamma counter (LKB). The data were analyzed by means of LIGflND fitting program (23).


Coronary uenular endothelial Endothelial cell membrane fragments

cells were





produced according to

(9). published

cultured our

was crosslinked to FGF receptors on the endothelial cell membrane fragments utilizing the reuersible crosslinker EGS. SDS-PRGE gels were run to identify the receptor molecule. The FGF was uncoupled from the receptor (please refer to methods) and the gel was transblotted to nitrocellulose. The receptor-containing portion of nitrocellulose was cut out and dissolued with acetonitrile. The SDS-PRGE purified receptor was either used as antigen in an in uitro immunization scheme (15) if primarily IgM antibodies were desired, or used to inject mice, the primed spleens of which were used for in uitro immunization with additional antigen, if primarily IgG classes of antibodies were desired. Membrane fragments were used in the ELISR and Western blot assays for the detection of anti-FGF receptor monoclonal antibodies (please refer to methods). Figure 1 shows the ELISR results for anti-FGF receptor clones UBSI and UBS2. When biotinylated sheep anti-mouse Ig (whole) was used as the second antibody during screening procedures, clones UBSI and UBS2 tested positiue for IgG antibody. Clone UBSI tested positiue when biotinylated goat anti-mouse IgM was used as the second antibody, showing that clone UBSI was secreting IgM antibodies. The production of IgM monoclonal antibody by clone UBSI was also demonstrated by isotyping (Figure 2).

protocol (13). lodinated FGF


Wh anti-mouse Ig


anti-mouse IgM

d Control

VBS -1


ELISR of monoclonal antibody producing clones UBSI and UBS2. FIGURE 1: Monoclonal antibody-producing clones UBSI and UBS2 tested positiue for anti-FGF receptor monoclonal antibodies when biotinylated sheep anti-mouse Ig (whole) was used as the second antibody. Clone UBSI tested positiue when biotinylated goat anti-mouse IgM was used as the second antibody, reuealing that UBSI secretes IgM antibody. reuealed that UBS2 secretes a mixture of IgG subclasses (data not also shows the elution profile of UBSI from the affinity purification column. Peak 1 (unbound fraction) fails to bind, while the majority of the material in peak 2 (bound fraction) binds to the unsolubilized Con-R column, indicating that the failure of peak 1 to bind, and the ability of peak 2 to bind, is not due to ouerloading of the column, or nonspecific absorption, respectiuely. The peak of purified IgM monoclonal antibody is indicated. Clone UBSI was loaded onto an IEF gel under non-reducing conditions. The transferred blot was probed with anti-IgG antibody (figure 3, lane B) and anti-IgM antibody (lane R) which confirmed that clone UBSI is an IgM monoclonal antibody. Isoelectrofocusing (IEF) shows that clone UBSI secretes monoclonal antibody possessing a pi of approximately 8.2 (Figure 3). Figure 4 shows that the molecular weight of the antibody under reducing conditions (SDS PAGE, 10% gel) is approximately 160 kDa, which agrees with the MW of an IqM subunit. The CUEC FGF receptor (11, 12) is a high affinity tyrosine kinase receptor. To further demonstrate that clone UBSI secretes an anti-FGF receptor monoclonal



Figure 2


Unbound fraction

0.2 M Mannoside

Bound fraction













FIGURE 2: Isotyping results and elution profile of antibody affinity purification of UBSI. Isotyping by a dipstick isotyping method (Rmersham) determined that UBSI secretes IgM antibody with a kappa light chain (insert). The elution profile of the Con-R affinity purification of monoclonal antibody UBSI is shown.


Pi 9.6

8.2 8.0 7.8 7.S 7.1 6.5

— —





FIGURE 3: Isoelectrofocusing gel of monoclonal antibody UBSI. Clone UBSI was loaded onto an IEF gel under non reducing conditions. Following IEF, the antibody was transblotted and probed with mouse anti-IgM antibody (1:2500) Lane R, and with mouse anti-IgG antibody (1:2000) Lane B. R streptauiden-HRP complex was then added (1:3000) and the reaction reuealed by enhanced chemiluminescence (ECL). Clone UBSI is shown to secrete IgM antibody possessing a pi of 8.2.





97.4 ·>



FIGURE 4: Molecular weight of monoclonal antibody UBSI subunit. Lane R: The molecular weight of the antibody (clone UBS 1) against the FGF receptor is ~160 kDa. Lane B: Positiue control (mouse IgM). Lane C: Negatiue control (normal goat


CUEC membrane preparations containing high affinity FGF receptors immunoprecipitated using anti-phosphotyrosine antibodies (please refer to methods). Figure 5, lane 1 shows the biotinylated high molecular weight standards. Lane 2 shows the CUEC membrane preparation that had been immunoprecipitated with anti-phosphytyrosine, electrophoresed, blotted onto nitrocellulose, and tested with culture supernatant from clone UBSI using

antibody, were

anti-mouse IgM as the second antibody. Monoclonal antibody UBSI detects the 120 kDa tyrosine kinase FGF receptor. In lane 3 mouse antiphosphotyrosine was used as the primary antibody followed by goat anti-mouse IgG. The tyrosine kinase receptor is indicated at 12B kDa, other phosphorylated molecules are also indicated. UBSI is capable of precipitating FGF receptor (data not shown), as well as detecting receptor immunoprecipitated by antiphosphotyrosine antibodies. The homologous displacement studies showed that bFGF is able to bind in this system with high affinity binding sites as shown by Scatchard analysis (Fig. 7) using binding buffer at pH 8.2. This pH was chosen because it has been shown that basic molecules can interact with the binding of FGF to its high and low affinity binding sites (24). By homologous binding studies on CUECs the receptors for bFGF was defined by a Kd of 75 pM and a capacity of 6.6 E-12 mol/l. Scatchard analysis of heterologous studies using UBSI and a control mouse IgM (Fig. 8) showed that mouse IgM did not displace the bFGF from its high affinity binding site on the receptor. UBSI was, howeuer, able to compete with bFGF for the receptor ligand binding site suggesting the efficacy of this antibody for the FGF receptor. Aeterologous binding studies showed that UBSI possessed a Kd of 152 pM and a capacity of 2.43E-I2 mol/l while the binding of FGF was estimated at a Kd of 46 pM and a capacity of 2.43E-12 mol/l. Monoclonal antibody UBSI works uery well for immunocytochemistry in chick, rat mouse, guinea pig, human, and bouine tissue (data not shown). Other species haue not yet been assayed.

biotinylated goat















FIGURE 5: Immunoprecipitation of FGF high affinity tyrosine kinase receptor followed by detection of the FGF receptor using anti-FGF receptor monoclonal antibody UBSI. Lane 1 Biotinylated high molecular weight standards. Lane 2 Membrane preparation from coronary uenular endothelial cells immunoprecipitated with rabbit anti-phosphotyrosine antibody, electrophoresed, transblotted to nitrocellulose, and tested with clone UBSI culture supernatant probing with biotinylated goat-anti-mouse IgM as the second antibody. Monoclonal antibody UBSI is shown to identify the phosphorylated FGF receptor at 120 kDa. Lane 3 Mouse anti-phosphotyrosine is used as the primary antibody following transblotting, goat anti-mouse IgG is the second antibody. The phosphorylated FGF receptor is indicated at 120 kDa, other phosphorylated molecules are indicated ( ·< ). -









Bound(M) FIGURE 6: Scatchard analysis of the competitiue binding studies of iodinated uersus cold bFGF. CUECs were incubated with 10 pM of '25l-bFGF with or without an isotopie dilution of unlabeled bFGF as reported in "Materials and methods". The data of the competitiue binding studies were analyzed by means of the LIGRND

fitting program.



Log 0.2





Bound(M) O •





Scatchard analysis of the heterologous competitiue and UBSI ( O ) uersus a control mouse IgM ( · ).


studies of


Rcidic and basic fibroblast

growth factors (FGFs)




the migration, proliferation and differentiation of endothelial cells to form blood uessels (25). These growth factors act through high affinity tyrosine kinase receptors to mediate their biological effects. Monoclonal antibodies against the FGF receptor may help define receptor epitopes inuolued in the FGF binding and signal transduction which mediate coronary and tumor angiogenesis. Commercially auailable polyclonal anti-FGF receptor antibodies are against receptor synthetic peptides, but not against the natiue receptor molecule. In uitro immunization has been found to be a suitable method for the preparation of anti-receptor monoclonal antibodies utilizing PRGE-purified receptor as the antigen. We haue preuiously shown the production of monoclonal antibodies against picogram quantities of antigen (15) and discussed the use of in uitro immunization for the production of monoclonal antibodies in response to



conserued molecules. We report here the production and characterization of monoclonal antibodies against the natiue FGF receptor molecule, rather than synthetic peptides, which may haue use in the study of functional receptor



Monoclonal antibody UBSI has been characterized as an IgM secreting clone and shown to be an exceptionally uersatile anti-FGF receptor monoclonal antibody of use in a uariety of assays. We haue demonstrated the usefulness of this

antibody in ELISR, Western blot, immunoprecipitation, and immunocytochemistry. Monoclonal antibody UBSI has been found to possess a subunit molecular weight of 160 kDa, and a pi of 8.2, both characteristic of IgM. The detection of a 120 kDa tyrosine kinase receptor by Western blot is well in range of the receptor molecular weight obtained by crosslinking ,25l-aFGF or ,25l-bFGF to the CUEC FGF receptor (10-12).

RCKNOWLEDGMENTS This research was supported by NIH HL41378 and Rmerican Heart Rffiliate grant WR-515, both awarded to MS.




1. 2.

3. 4.


6. 7.

8. 9. 10. 11.




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reprint requests to: M. E.


Department of Genetics & Cell Biology Washington State Uniuersity Pullman, WR 99164-4234 Received for publication: 3/17/92 after revision: 8/11/92



Production of anti-fibroblast growth factor receptor monoclonal antibodies by in vitro immunization.

Monoclonal antibodies against the high affinity tyrosine kinase Fibroblast Growth Factor (FGF) receptor may help define receptor epitopes involved in ...
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