Production of anti-carbohydrate antibodies by phage display technologies: potential impairment of cell growth as a result of endogenous expression.
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 IgG(1) 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-Le(x) phages were screened from the same library with lacto-N-fucopentaose III (LNFPIII; Le(x))-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.
Yuasa
Noriyuki
N
Department of Applied Biochemistry, Tokai University School of Engineering, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan.
Zhang
Wei
W
Goto
Tomohiro
T
Sakaue
Hiroyuki
H
Matsumoto-Takasaki
Ayano
A
Kimura
Miyo
M
Ohshima
Hiroya
H
Tsuchida
Yasunobu
Y
Koizumi
Tomoyuki
T
Sakai
Keiko
K
Kojima
Takumi
T
Yamamoto
Kazuo
K
Nakata
Munehiro
M
Fujita-Yamaguchi
Yoko
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D051379:Mice; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D014312:Trisaccharides / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D057127:Single-Chain Antibodies; D014312:Trisaccharides; C023872:mannotriose