Production of Anti-carbohydrate Antibodies by Phage Display Technologies

• Published in: The Journal of biological chemistry 2010-10, Vol.285 (40), p.30587-30597
• Main Authors: Yuasa, Noriyuki, Zhang, Wei, Goto, Tomohiro, Sakaue, Hiroyuki, Matsumoto-Takasaki, Ayano, Kimura, Miyo, Ohshima, Hiroya, Tsuchida, Yasunobu, Koizumi, Tomoyuki, Sakai, Keiko, Kojima, Takumi, Yamamoto, Kazuo, Nakata, Munehiro, Fujita-Yamaguchi, Yoko
• Format: Article
• Language: English
• Subjects: Anti-carbohydrate Antibodies; Antibodies; Antibody Production; Carbohydrate; Carbohydrate-binding Protein; ER Stress; Glycobiology; Glycoprotein Biosynthesis; Phage Display; Single Chain antibody
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.107284

Anti-mannotriose (Man3) antibodies were previously isolated from a Keio phage library displaying human single chain variable fragments (scFvs) using a neoglycolipid, Man3- dipalmitoylphosphatidylethanolamine. Of three genes constructed, the 5A3 clone was expressed in mouse myeloma NS0 cells as a conjugate with human IgG1 Fc (scFv-Fc) and characterized (Sakai, K., Shimizu, Y., Chiba, T., Matsumoto-Takasaki, A., Kusada, Y., Zhang, W., Nakata, M., Kojima, N., Toma, K., Takayanagi, A., Shimizu, N., and Fujita-Yamaguchi, Y. (2007) Biochemistry 46, 253–262; Zhang, W., Matsumoto-Takasaki, A., Kusada, Y., Sakaue, H., Sakai, K., Nakata, M., and Fujita-Yamaguchi, Y. (2007) Biochemistry 46, 263–270). Similarly, anti-Lex phages were screened from the same library with lacto-N-fucopentaose III (LNFPIII; Lex)-dipalmitoylphosphatidylethanolamine. Of five phage clones isolated, two scFv genes were constructed to express scFv-Fc proteins in NS0 cells. As was experienced with anti-Man3 scFv-Fc clones, only one anti-LNFPIII clone, 1F12, was successfully produced and purified as an scFv-Fc protein. Although anti-LNFPIII 1F12 and anti-Man3 5A3 scFv-Fc proteins were secreted into media, a decline in scFv-Fc production was observed with both stable clones during early passages. Transient expression of anti-LNFPIII and anti-Man3 scFv-Fc genes in COS-7 cells and subsequent analyses of scFv-Fc protein expression revealed accumulation of translated proteins in the endoplasmic reticulum for scFv-Fc proteins derived from clones that did not survive as stable clones. This report describes the following: (i) isolation of anti-LNFPIII scFv genes; (ii) purification of anti-LNFPIII scFv-Fc proteins from stably and transiently expressed cells; and (iii) extracellular or intracellular localization of two anti-LNFPIII and three anti-Man3 scFv-Fc proteins. The results suggest that expression of anti-Man3 and other anti-carbohydrate antibodies in mammalian cells is disadvantageous for cell growth.