Production of Anti-carbohydrate Antibodies by Phage Display Technologies: POTENTIAL IMPAIRMENT OF CELL GROWTH AS A RESULT OF ENDOGENOUS EXPRESSION* |
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| Authors: | Noriyuki Yuasa Wei Zhang Tomohiro Goto Hiroyuki Sakaue Ayano Matsumoto-Takasaki Miyo Kimura Hiroya Ohshima Yasunobu Tsuchida Tomoyuki Koizumi Keiko Sakai Takumi Kojima Kazuo Yamamoto Munehiro Nakata Yoko Fujita-Yamaguchi |
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| Institution: | From the ‡Department of Applied Biochemistry, Tokai University School of Engineering, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan and ;the §Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa 277-8562, Japan |
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| Abstract: | 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. |
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| Keywords: | Antibodies Carbohydrate Carbohydrate-binding Protein ER Stress Glycoprotein Biosynthesis Glycobiology Anti-carbohydrate Antibodies Antibody Production Phage Display Single Chain antibody |
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