ANALYTICAL

BIOCHEMISTRY

188,

%‘-1oo

(1990)

Nonradioactive Ligand Bindi ng Assay for Epidermal Growth Factor Receptor Ivan C. King and Joseph J. Catino’ Department of Experimental Therapeutics, Bristol-P Myers Squibb Company, 5 Research Parkway,

Received

March

5,199O

A rapid and sensitive nonradioactive ligand binding assay for identifying the agonists and antagonists of epidermal growth factor receptor is described. The assay is easy to perform, has a within-assay error of less than 15%, and does not use radioactive substances. Nonspecific binding is less than 10% and the assay is sensitive enough to detect epidermal growth factor (EGF) antagonists at as little as 10 nM, if the antagonist has the same affinity as EGF. The assay is carried out in 96-well plates and is particularly suitable for largescale screening. This report includes description of the methods that we used to assess the validity of the assay system, and the results of that assessment. o 1990 Academic

Press,

Inc.

The polypeptide EGF,’ originally isolated from the submaxillary gland of mice, is mitogenic for various types of cells (1). EGF has been identified in numerous tissues and body fluids of many mammalian species (2). Proteins that bear homology to EGF include blood clotting factors IX and X, protein C, plasminogen activator, vaccinia growth factor, and TGF-a (3). Proteins with similarities to EGF have also been found in lower orders of organisms, such as Caenorhabditis elegans and DFOsophila (4). A potent agonist for the EGF receptor, identified in spontaneous tumors and in cells that have been transformed by exposure to retrovirus, is identified as TGF-a (5). A patient who bears a tumor commonly has elevated levels of TGF-a in body fluids (6). Transfection of FR3T3 fibroblasts with a synthetic oligonucleotide that encodes for EGF induces cellular transformation (7). ’ Current address: Department of Tumor Biology, Schering-Plough Research, 60 Orange Street, Bloomfield, NJ 07003. ’ Abbreviations used: B-EGF, biotinylated EGF; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; EGF, epiderma1 growth factor; FBS, fetal bovine serum; PBS, phosphate-buffered saline; TGF-cu, transforming growth factor-a. 0003-2697/90 Copyright All rights

Wallingford, Connecticut 06492

$3.00 0 1990 by Academic Press, of reproduction in any form

The EGF receptor is a 170-kDa glycoprotein that has, at its c-terminus, tyrosine-specific protein kinase activity. An oncogene (verb B) isolated from the avian erythroblastosis virus has regions of homology with the EGF receptor (8). Binding of EGF to the receptor activates the protein kinase to phosphorylate several cellular proteins, such as lipocortin I and S6 kinase (9). Certain carcinoma cells bear an excess of EGF receptors; several reports have affirmed that squamous cell carcinomas bear 2.5 to 55 times more EGF receptors than do normal epidermal cells (10). Such amplification or overexpression of the EGF receptor has been detected in gliomas, and in bladder and esophageal carcinomas. The overexpression of the EGF receptor in NIH-3T3 cells increases the anchorage-independent growth in soft agar, and adding EGF or TGF-a will enhance this phenomenon. Furthermore, Velu et al. (11) reported that cells transfected by the EGF receptor will produce tumor in nude mice, whereas nontransfected cells will not produce tumor. These results support the hypothesis of autocrine-directed cell growth, which was originally based upon the discovery of TGF-(U (12). We are interested in studying factor-like substances from transformed neoplastic cells to evaluate their role in growth of the epidermally derived cells. The ability of neoplastic cells to secrete peptides that render tumor cell proliferation independent of exogenous factors has been demonstrated (13). An agent, such as an EGF antagonist, that is capable of competing with the growth factor might be useful in the treatment of some types of carcinoma. In this report, a simple and sensitive method that does not use radioactive materials is described for identifying EGF receptor antagonists and agonists. MATERIALS

AND

METHODS

Cell culture. Human epidermal A431 cells (American Type Culture Collection, Rockville, MD) were propagated in equal volumes of Dulbecco’s modified Eagle’s medium (DMEM) and Ham F12 medium supplemented 97

Inc. reserved.

98

KING

AND

CATINO

10% with fetal bovine serum. Hybridoma cells were developed in our laboratory by fusion with spleen cells from mice immunized with A431 cells. These hybridomas were the source of anti-EGF receptor antibodies designated Ab-3 and Ab-4; Ab-1 and Ab-2 were purchased (Oncogene Science, Manhasset, NY). Biotinylation of EGF. EGF (Biomedical Technologies, Stoughton, MA) was dissolved to 1 mg/ml with 0.1 M sodium bicarbonate (pH 8.5). To this solution, 1.12 mg of biotin-N-hydroxycaproicsuccinimide (Sigma Chemical Co., St. Louis, MO) in 20 ~1 of dimethyl sulfoxide was added and the solution was incubated at 25°C for 4 h. The reaction was stopped by addition of 100 ~1 of 2 M glycine and the mixture was dialyzed against PBS at 4°C overnight. Assay of biotinylated EGF. The coupling efficiency of the biotin with EGF was assessed by the standard lz51EGF ligand binding assay. A portion of the dialyzed mixture was filtered through an avidin-agarose column (Vector Laboratories, Burlingame, CA) to remove the BEGF; unfiltered and filtered mixtures were analyzed. The ratio of the EGF binding activity of the unfiltered to the filtered mixture indicates the efficiency of EGF coupling to the biotin. Competitive binding of B-EGF and EGF. For 24 h before the assay, A431 cells (105/well) were seeded into 24well plates. The method of Green and Couchman (14) was used to prepare 1251-EGF for the assay. The procedure for the assay was the same as that described (15), except that B-EGF was included at various concentrations. Surface EGF receptor detected by B-EGF. For 24 h before the assay, A431 cells (l-5 X 104/well) were grown in 96-well plates. Cells washed in the wells twice with PBS were exposed to 0.5% formaldehyde for 15 min at room temperature. The fixed cells, washed three times with PBS, were incubated in 3% FBS at 37°C for 30 min. After the FBS was removed, B-EGF at various concentrations in DMEM supplemented with 0.1% BSA and 20 mM Hepes (pH 7.4) was added and the plates were incubated at 37°C for 60 min. In this step, the agents being evaluated for binding to the EGF receptor were included; the total volume was 50 ~1. After incubation, the cells were washed four times with PBS that contained 0.3 M NaCl. After incubation with streptavidinconjugated horseradish peroxidase (Zymed Laboratories, San Francisco, CA) at 37°C for 1 h, the cells were washed with PBS and 0.3 M NaCl. The washed cells were exposed to 2,2’-azido-di[3-ethylbenzthiazoline-6sulfonic acid] and hydrogen peroxide (Bio-Rad, Richmond, CA) for 25 min; this reacts with the horseradish peroxidase, to the extent it was bound to the cells, to produce a colored product. The presence of the colored product was assessedby absorbance measurement in an automatic reader for 96-well plates, at a 410-nm wave-

04 I

7

10 E(K

lnghel

30

100

1)

FIG. 1. Displacement of lz51-EGF by B-EGF EGF was added to various concentrations (l-100 beled EGF; (m) B-EGF.

or EGF. rig/well).

B-EGF or (+) Unla-

length; nonspecific binding was assessedby adding 1 pg/ well of EGF. Statistical analysis. The mean f. standard deviation of at least three different experiments, each of which carried out in triplicates, was presented unless specified. RESULTS

AND

DISCUSSION

The method used conjugates one or more molecules of biotin to nearly every molecule of epidermal growth factor. Although the ratio of biotin coupling to EGF has not been evaluated for the reaction, the high ratio of BEGF retained on the avidin column compared to unlabeled EGF (data not shown) suggeststhat nearly all the EGF was conjugated to biotin. Each batch of B-EGF was evaluated for its ability to bind to the EGF receptor. BEGF was found to exhibit a binding affinity to the EGF receptor similar to that of the unlabeled EGF, as measured by the radioactive ligand binding assay (Fig. 1). Procedures that might affect the B-EGF binding assay, such as fixative and wash conditions, were extensively evaluated. We found that cells fixed with 0.5% formaldehyde for 15 min provided the greatest sensitivity and the least nonspecific binding, compared to cells prepared by other techniques. Figure 2 represents the binding of B-EGF to A431 cells in the assay; specific binding was saturable and nonspecific binding was always less than 10% of the total binding. The binding activity increased with increasing number of cells in the well, reaching a plateau between 4 X lo4 and 5 X lo4 cells/well (Fig. 3). As expected, added B-EGF increased the amount of binding activity. The results of assessment of the competitive binding among B-EGF, biotin, and EGF are graphically presented in Fig. 4. These results show that unlabeled EGF did compete with the B-

NONRADIOACTIVE 0.6

LIGAND

BINDING

T I

0.0”

0.156

ASSAY

FOR

EPIDERMAL

GROWTH

RECEPTOR

4

e

0.312

0.625

1.25

2.5

0

5

4

1

2

tog/well1

Biotin

FIG. 2. Binding curve of various amounts of B-EGF to A431 cells (3 X lo4 cells/well) in 96-well dishes. B-EGF was added to various concentrations (0.156-5 rig/well) to determine the specific binding activity of B-EGF to the EGF receptor. The results represent the means + SD of triplicate samples in a typical experiment.

EGF for the receptors, but that biotin did not compete, indicating that the binding of the B-EGF is mediated through the EGF rather than through the biotin. The I(&, for EGF in this assay is about 3 rig/well (at 1.25 rig/well of B-EGF); i.e., the assay is able to detect EGF antagonists or agonists at a concentration of 10 nM or less if they have the same affinity for the receptor as that possessed by the EGF. We have also assessed various growth factors for competition in binding the EGF receptor. The data in Table

1 show that the only factors capable of competing with B-EGF are those that are related to EGF. In this assay, TGF-a was as potent as EGF, whereas neither insulin nor transferrin effectively competed with B-EGF for the receptor. Many antibodies with specificity for the EGF receptors can inhibit the binding of EGF to its receptors (16). In this assay, we showed that Ab-1, Ab-2, and Ab3 had EGF antagonist activities; the binding affinity of

0.5’-

Treatments

Concentration b.cdml)

EGF

d d

1 0.25 0.06 0.25 5 5 15 3.75 0.94 15 3.75 0.94 300 75 18.8 720

0.P-

TGF-ol Insulin Transferrin Ab-1

0.2--

1.0

2.0

Cell

3.0

number

4.0

I 5.0

Ab-2

(xl(r4)

FIG. 3. Binding of B-EGF to various numbers of A431 cells. Various concentrations of B-EGF (1.25, 2.5, and 5.0 rig/well) were added to plates seeded with various numbers of cells (1.0-5.0 X 104/well). (u) 5 rig/well; (0) 2.5 rig/well; (A) 1.25 rig/well. The mean of triplicate samples in a typical experiment is presented. The standard deviation is less than 15% of the mean in every sample.

1

Competition Binding between B-EGF and Other Growth Factors and Antibodies

4

0.4--

32

hg/well)

OP EGF

TABLE I

16

FIG. 4. Competition between B-EGF and EGF or biotin for EGF receptors. B-EGF was added to 1.25 or 2.5 rig/well, either alone or in combination: (w) B-EGF at 2.5 rig/well with biotin; (0) B-EGF at 1.25 rig/well with biotin; (0) B-EGF at 2.5 rig/well with EGF, (0) B-EGF at 1.25 rig/well with EGF.

0.6

0

99

T

G-EGF

0.i-l

FACTOR

Ab-3

Ab-4 a Zero represents

no inhibition

or even an increase

% Inhibition (range) 94.3 88.3 79.6 89.0 on 0 98.3 92.0 71.1 64.1 62.0 29.0 80.1 68.5 13.3 5.9

(98.7-90.9) (92.9-84.2) (85.6-70.8) (92.9-87.0) (7.4-O) (4.5-O) (100-96.0) (93.1-90.9) (79.6-60.6) (69.3-54.4) (67.2-50.9) (36.9-O) (85.2-76.0) (77.1-55.5) (16.2-8.4) (7.0-4.1)

in binding.

100

KING

AND

Ab-1 appears to have been greater than those of Ab-2 and Ab-3. The procedure for the B-EGF binding assay is simple, is low in cost, and does not use radioactive materials and the variation for replicates within an assay is small, always less than 15%. The assay uses 96-well plates that render it especially useful for the purpose of large-scale screening. This assay has been used for the screening of over 1000 crude fermentation samples and the data generated so far confirmed the feasibility of using this assay for identifying EGF receptor antagonists. We are currently using the B-EGF binding assay to examine active components purified from the fermentation samples identified by this assay. REFERENCES 1. Cohen,

S. (1962)

CATINO

5. De Larco, USA

J. E., and Todaro, 75,4001-4005.

Chem.

237,1555-1562.

2. Mattila, A.-L., Saario, I., Viinikka, L., Ylikorkala, O., and Perheentupa, J. (1988) Brit. J. Cancer 67,139-141. 3. Appella, E., Weber, I. T., and Blasi, F. (1988) FEBS Lett. 231, l-4. 4. Greenwald, I. (1988) BioEssays 6,70-73.

Proc.

N&l.

Acad.

Sci.

6. Arteaga,

C. L., Hanauske, A. R., Clark, G. M., Osborne, C. K., Hazarika, P., Pardue, R. L., Tio, F., and Von Hoff, D. D. (1988) Cancer Res. 48,5023-5028.

7. Stern,

D. F., Hare, 235,321-325.

Science

D. L., Cecchini,

8. Downward,

J., Yarden, Y., Mayes, well, P., Ullrich, A., Schlessinger, Nature (London) 307,521-527.

9. Fava,

R. A., and Cohen,

S. (1984)

M. A., Weinberg,

R. A. (1987)

C., Scrace, G., Totty, N., StockJ., and Waterfield, M. D. (1984) J. Biol.

Chem.

259,2636-2645.

10. Gullick, W. J., Marsden, J. J., Whittle, N., Ward, B., Bobrow, and Waterfield, M. D. (1986) Cancer Res. 46,414-416.

L.,

11. Velu, T. J., Beguinot, L., Vass, W. C., Willingham, M. C., Merlino, G. T., Pastan, I., and Lowy, D. R. (1987) Science 238,1408-1410. 12. Sporn, M. B., and Todaro, G. J. (1980) N. Engl. J. Med. 303,878880. 13. Salomon,

J. Biol.

G. J. (1978)

D. S., and Perroteau,

I. (1986)

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14. Green, M. R., and Couchman, 239-245.

J. R. (1985)

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86,

15. King, I. CL., and Sartorelli, Commun. 140,837-843.

A. C. (1986)

Biochem.

Biophys.

Res.

16. Kawamoto, T., Sato, J. D., Le, A., Polikoff, J., Sato, G., and Mendelsohn, J. (1983) Proc. N&l. Acad. Sci. USA 80.1337-1341.