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The efficient production of monoclonal antibodies to the epidermal growth factor receptor
Journal of Immunological Methods, 103 (1987) 153-154
153
Elsevier JIM 04572
Letter to the editors
The efficient production of monoclonal antibodies to the epidermal growth factor receptor Ivan King and Alan C. Sartorelli Department of Pharmacology and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT06510, U.S.A. (Received 15 June 1987, revised received 20 July 1987, accepted 28 July 1987)
Dear Editors, The production of monoclonal antibodies (MAbs) by the methodology developed by KShler and Milstein (1975) involves the production of hybridomas through the fusion of myeloma cells with spleen cells isolated from immunized animals. A variety of immunization schedules have been reported; most of these approaches require multiple injections of immunogen over a period of 4-8 weeks. The procedures are not only time-consuming, but also require a relatively large quantity of antigen. Spitz et al. (1984) have described an immunization procedure to generate MAbs by the intrasplenic injection of antigen. This procedure has been reported to markedly shorten the period of immunization and to reduce significantly the amount of antigen required, the availability of sufficient quantities of pure antigen for the immunization and screening procedures often being a significant obstacle to the routine generation of MAbs. To accomplish the cloning of hybridomas, a variety of feeder cells, including thymocytes and macrophages, have been used. The presence of growth factor supplements has been reported to support the clonal growth of hybridomas in the absence of a feeder layer of cells (Pintus et al., 1983), the utilization of such supplementation serving to facilitate the process of MAb produc-
Correspondence to: A.C. Sartorelli, Department of Pharmacology and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT 06510, U.S.A.
tion. Antibodies have been made against synthetic peptides using sequences that are part of the protein of interest. Thus, synthetic peptides can serve as a reliable source of immunogen for the production of MAbs. We have successfully used a synthetic peptide to produce MAbs that recognize the intact epidermal growth factor receptor (EGFR) within 1 month, including the time required to prepare the immunogen. The antigen employed for the immunization procedure was a dodecapeptide whose sequence corresponded to amino acid residues 985-996 of the EGFR (Gullick et al., 1985). The peptide was conjugated to keyhole limpet hemocyanine by the addition of glutaraldehyde. The intrasplenic immunization procedure of Spitz et al. (1984) was used to produce MAbs; a single injection of 15 or 100/~g of conjugated peptide in 100/~1 of PBS was used for the immunization. Non-secreting P3X63Ag8.653 mouse myeloma cells were used for the cell fusion. Cells were routinely maintained in RPMI 1640 medium supplemented with 20% fetal bovine serum, 2 mM glutamine, 100 U / m l of penicillin, and 100 /~g/ml of streptomycin. The fusion procedure was basically employed in the manner described by Galfr6 et al. (1977). Supernatants were screened for the presence of specific antibody using an enzyme-linked immunosorbent assay. Hybridomas secreting specific antibodies were seeded in culture medium supplemented with 2% (v/v) Ewing sarcoma growth factor (ESGF; ICN Biochemicals, Cleveland, OH, and Costar Europe, Badhoevedorp, The Netherlands) at densities of 0.6, 2 and 6 cells/well in 200 /~1 of
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
154 medium. Supernatants were assayed for specific antibodies after 7 - 1 4 days. Analysis of the E G F R was accomplished by labeling A431 cells with [3Zp]orthophosphate followed by precipitation of the receptor by the addition of M A b . The i m m u n e complexes were analyzed by electrophoresis on 7.5% SDS-polyacrylamide gels. After electrophoresis, proteins were visualized by either Coomassie blue or silver staining. The gels were then dried and exposed to X-ray film at - 7 0 o C. Using intrasplenic immunization, we f o u n d that more I g M than I g G antibodies were consistently produced. Thus, antibody typing performed on hybridomas after cloning showed that > 90% of the clones secreted I g M antibodies. M A b s were successfully generated even when animals were immunized with a single intrasplenic injection of only 15/~g of EGFR-specific peptide. L y m p h o i d cells do not grow well at low densities and feeder cells or growth factors are always required for the expansion and cloning of hybridomas. The utilization of feeder cells has a n u m b e r of disadvantages, including the possibility of contamination and the continuous need to maintain a supply of animals to provide these cells. We have found that E S G F , m a n u f a c t u r e d by two different commercial sources, was capable of supporting the cloning of hybridomas. The cloning efficiency using E S G F was comparable to that obtained using macrophages as feeder cells. Furthermore, most hybrids obtained after cloning with the supplementation of E S G F were able to secrete specific MAbs. Three M A b s made against a dodecapeptide corresponding to amino acid residues 9 8 5 - 9 9 6 of the E G F R were able to recognize and precipitate the intact receptor. Thus, using a synthetic peptide as i m m u n o g e n and a novel immunization schedule, it was possible to obtain M A b s within a period as short as one m o n t h after the sequence of the protein of interest was determined.
The E G F R can be divided into two domains: an amino terminus of 621 amino acid residues with E G F binding activity, and a carboxyl terminus of 542 amino acids with protein kinase activity. It has been shown that m a n y tumors produce truncated E G F R s , with either the E G F binding portion or the tyrosine kinase domain being deleted (Lin et al., 1984; H u m p h r e y et al., 1987). The production of M A b s that recognize either the extracellular or the intracellular d o m a i n (such as the M A b s reported herein) should be useful in the elucidation of the role of the truncated receptor in malignant transformation.
References Galfr& G., Howe, S.C., Milstein, C., Butcher, G.W. and Howard, J.C. (1977) Antibody to major histocompatibility antigens produced by hybrid cell lines. Nature 266, 550. Gullick, W.J., Downward, J. and Waterfield, M.D. (1985) Antibodies to the autophosphorylaton sites of the epidermal growth factor receptor protein kinase as probes of structure and function. EMBO J. 4, 2869. Humphrey, P.A., Wong, A.J., Friedman, H.S., Werner, M.H., Vogelstein, B., Bigner, D.D. and Bigner, S.H. (1987) Amplification and expression of the epidermal growth factor receptor (EGFR) gene in human glioma (HGL) xenografts. Proc. Am. Assoc. Cancer Res. 28, 23. K/Shier, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495. Lin, C.R., Chen, W.S., Kruiger, W., Stolarsky, L.S., Weber, W.. Evans, R.M., Verma, I.M., Gill, G.N. and Rosenfeld, M.G. (1984) Expression cloning of human EGF receptor complementary DNA: Gene amplification and three related messenger RNA products in A431 cells. Science 224, 843. Pintus, C., Ransom, J.H. and Evans, C.H. (1983) Endothelial cell growth supplement: A cell cloning factor that promotes the growth of monoclonal antibody producing hybridoma cells. J. Immunol. Methods 61,195. Spitz, M., Spitz, L., Thorpe, R. and Eugui, E. (1984) lntrasplenic primary immunization for the production of monoclonal antibodies. J. Immunol. Methods 70, 39.
153
Elsevier JIM 04572
Letter to the editors
The efficient production of monoclonal antibodies to the epidermal growth factor receptor Ivan King and Alan C. Sartorelli Department of Pharmacology and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT06510, U.S.A. (Received 15 June 1987, revised received 20 July 1987, accepted 28 July 1987)
Dear Editors, The production of monoclonal antibodies (MAbs) by the methodology developed by KShler and Milstein (1975) involves the production of hybridomas through the fusion of myeloma cells with spleen cells isolated from immunized animals. A variety of immunization schedules have been reported; most of these approaches require multiple injections of immunogen over a period of 4-8 weeks. The procedures are not only time-consuming, but also require a relatively large quantity of antigen. Spitz et al. (1984) have described an immunization procedure to generate MAbs by the intrasplenic injection of antigen. This procedure has been reported to markedly shorten the period of immunization and to reduce significantly the amount of antigen required, the availability of sufficient quantities of pure antigen for the immunization and screening procedures often being a significant obstacle to the routine generation of MAbs. To accomplish the cloning of hybridomas, a variety of feeder cells, including thymocytes and macrophages, have been used. The presence of growth factor supplements has been reported to support the clonal growth of hybridomas in the absence of a feeder layer of cells (Pintus et al., 1983), the utilization of such supplementation serving to facilitate the process of MAb produc-
Correspondence to: A.C. Sartorelli, Department of Pharmacology and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT 06510, U.S.A.
tion. Antibodies have been made against synthetic peptides using sequences that are part of the protein of interest. Thus, synthetic peptides can serve as a reliable source of immunogen for the production of MAbs. We have successfully used a synthetic peptide to produce MAbs that recognize the intact epidermal growth factor receptor (EGFR) within 1 month, including the time required to prepare the immunogen. The antigen employed for the immunization procedure was a dodecapeptide whose sequence corresponded to amino acid residues 985-996 of the EGFR (Gullick et al., 1985). The peptide was conjugated to keyhole limpet hemocyanine by the addition of glutaraldehyde. The intrasplenic immunization procedure of Spitz et al. (1984) was used to produce MAbs; a single injection of 15 or 100/~g of conjugated peptide in 100/~1 of PBS was used for the immunization. Non-secreting P3X63Ag8.653 mouse myeloma cells were used for the cell fusion. Cells were routinely maintained in RPMI 1640 medium supplemented with 20% fetal bovine serum, 2 mM glutamine, 100 U / m l of penicillin, and 100 /~g/ml of streptomycin. The fusion procedure was basically employed in the manner described by Galfr6 et al. (1977). Supernatants were screened for the presence of specific antibody using an enzyme-linked immunosorbent assay. Hybridomas secreting specific antibodies were seeded in culture medium supplemented with 2% (v/v) Ewing sarcoma growth factor (ESGF; ICN Biochemicals, Cleveland, OH, and Costar Europe, Badhoevedorp, The Netherlands) at densities of 0.6, 2 and 6 cells/well in 200 /~1 of
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
154 medium. Supernatants were assayed for specific antibodies after 7 - 1 4 days. Analysis of the E G F R was accomplished by labeling A431 cells with [3Zp]orthophosphate followed by precipitation of the receptor by the addition of M A b . The i m m u n e complexes were analyzed by electrophoresis on 7.5% SDS-polyacrylamide gels. After electrophoresis, proteins were visualized by either Coomassie blue or silver staining. The gels were then dried and exposed to X-ray film at - 7 0 o C. Using intrasplenic immunization, we f o u n d that more I g M than I g G antibodies were consistently produced. Thus, antibody typing performed on hybridomas after cloning showed that > 90% of the clones secreted I g M antibodies. M A b s were successfully generated even when animals were immunized with a single intrasplenic injection of only 15/~g of EGFR-specific peptide. L y m p h o i d cells do not grow well at low densities and feeder cells or growth factors are always required for the expansion and cloning of hybridomas. The utilization of feeder cells has a n u m b e r of disadvantages, including the possibility of contamination and the continuous need to maintain a supply of animals to provide these cells. We have found that E S G F , m a n u f a c t u r e d by two different commercial sources, was capable of supporting the cloning of hybridomas. The cloning efficiency using E S G F was comparable to that obtained using macrophages as feeder cells. Furthermore, most hybrids obtained after cloning with the supplementation of E S G F were able to secrete specific MAbs. Three M A b s made against a dodecapeptide corresponding to amino acid residues 9 8 5 - 9 9 6 of the E G F R were able to recognize and precipitate the intact receptor. Thus, using a synthetic peptide as i m m u n o g e n and a novel immunization schedule, it was possible to obtain M A b s within a period as short as one m o n t h after the sequence of the protein of interest was determined.
The E G F R can be divided into two domains: an amino terminus of 621 amino acid residues with E G F binding activity, and a carboxyl terminus of 542 amino acids with protein kinase activity. It has been shown that m a n y tumors produce truncated E G F R s , with either the E G F binding portion or the tyrosine kinase domain being deleted (Lin et al., 1984; H u m p h r e y et al., 1987). The production of M A b s that recognize either the extracellular or the intracellular d o m a i n (such as the M A b s reported herein) should be useful in the elucidation of the role of the truncated receptor in malignant transformation.
References Galfr& G., Howe, S.C., Milstein, C., Butcher, G.W. and Howard, J.C. (1977) Antibody to major histocompatibility antigens produced by hybrid cell lines. Nature 266, 550. Gullick, W.J., Downward, J. and Waterfield, M.D. (1985) Antibodies to the autophosphorylaton sites of the epidermal growth factor receptor protein kinase as probes of structure and function. EMBO J. 4, 2869. Humphrey, P.A., Wong, A.J., Friedman, H.S., Werner, M.H., Vogelstein, B., Bigner, D.D. and Bigner, S.H. (1987) Amplification and expression of the epidermal growth factor receptor (EGFR) gene in human glioma (HGL) xenografts. Proc. Am. Assoc. Cancer Res. 28, 23. K/Shier, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495. Lin, C.R., Chen, W.S., Kruiger, W., Stolarsky, L.S., Weber, W.. Evans, R.M., Verma, I.M., Gill, G.N. and Rosenfeld, M.G. (1984) Expression cloning of human EGF receptor complementary DNA: Gene amplification and three related messenger RNA products in A431 cells. Science 224, 843. Pintus, C., Ransom, J.H. and Evans, C.H. (1983) Endothelial cell growth supplement: A cell cloning factor that promotes the growth of monoclonal antibody producing hybridoma cells. J. Immunol. Methods 61,195. Spitz, M., Spitz, L., Thorpe, R. and Eugui, E. (1984) lntrasplenic primary immunization for the production of monoclonal antibodies. J. Immunol. Methods 70, 39.