title: Immunization method for multi-pass membrane proteins using highly metastatic cell lines.
abstract: A novel method using metastatic breast cancer cell lines was established for producing monoclonal antibodies (mAbs) against multi-span membrane proteins. Grafting of metastatic cells (MCF7-14) into the mammary gland of BALB/cJ/nu/nu mice induced splenic hypertrophy (1.6-3.0×10(8)cells/spleen [n=6]). More than half of the mAbs against MCF7-14 cells reacted with the cell membrane. Inducing production of antibodies against the extracellular domain of multi-pass membrane proteins is difficult. Because the protein structure becomes more complex as the number of transmembrane domains increases, preparing antigens for immunization in which the original structure is maintained is challenging. Using highly metastatic MDA-MB231 cells as the host cell line, we produced mAbs against a 12 transmembrane protein, solute carrier family 6 member 6 (SLC6A6), as a model antigen. When SLC6A6-overexpressing MDA-MB231 cells were grafted into nude mice, the number of splenocytes increased to 2.7-11.4×10(8)cells/spleen (n=10). Seven mAb-producing clones that not only recognized the extracellular domain of SLC6A6 but also were of the IgG subclass were obtained. Immunocytochemistry and flow cytometry analyses revealed that these mAbs recognized the native form of the extracellular domain of SLC6A6 on the cell surface. Our novel immunization method involving highly metastatic cells could be used to develop therapeutic mAbs against other multi-pass membrane proteins.
authors.affiliation: Research and Development Division, Order-made Medical Research Inc., A-18 Green House, 2639 Yamazaki, Noda, Chiba 278-0022, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Satofuka
authors.affiliation: Research and Development Division, Order-made Medical Research Inc., A-18 Green House, 2639 Yamazaki, Noda, Chiba 278-0022, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Okabe
authors.affiliation: Department of Biological Science and Technology, Graduate School of Industrial Science and Technology, Tokyo University of Science, 3-1, Niijyuku 6-chome, Katsushika-ku, Tokyo 125-8585, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Takano
authors.affiliation: Research and Development Division, Order-made Medical Research Inc., A-18 Green House, 2639 Yamazaki, Noda, Chiba 278-0022, Japan.
authors.forename: Takahiko
authors.initials: T
authors.lastname: Utsugi
authors.affiliation: Department of Biological Science and Technology, Graduate School of Industrial Science and Technology, Tokyo University of Science, 3-1, Niijyuku 6-chome, Katsushika-ku, Tokyo 125-8585, Japan.
authors.forename: Masaya
authors.initials: M
authors.lastname: Ohtsu
authors.affiliation: Research and Development Division, Order-made Medical Research Inc., A-18 Green House, 2639 Yamazaki, Noda, Chiba 278-0022, Japan; Department of Biological Science and Technology, Graduate School of Industrial Science and Technology, Tokyo University of Science, 3-1, Niijyuku 6-chome, Katsushika-ku, Tokyo 125-8585, Japan. Electronic address: yasufumi@rs.noda.tus.ac.jp.
authors.forename: Yasufumi
authors.initials: Y
authors.lastname: Murakami
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002460:Cell Line; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D007114:Immunization / Q000379:methods*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D008565:Membrane Proteins / Q000276:immunology*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009362:Neoplasm Metastasis / Q000276:immunology*; D009363:Neoplasm Proteins / Q000276:immunology*; D015202:Protein Engineering / Q000379:methods*; D000099247:Taurine Transporters
chemical_list: D000911:Antibodies, Monoclonal; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009363:Neoplasm Proteins; D000099247:Taurine Transporters
keywords: Breast cancer; Immunization method; Metastasis; Mouse monoclonal antibody