Characterization of N-glycan structures and biofunction of anti-colorectal cancer monoclonal antibody CO17-1A produced in baculovirus-insect cell expression system.
Advantages of the baculovirus insect cell expression system for production of recombinant proteins include high capacity, flexibility, and glycosylation capability. In this study, this expression system was exploited to produce anti-cancer monoclonal antibody (mAb) CO17-1A, which recognizes the antigen GA733. The heavy chain (HC) and light chain (LC) genes of mAb CO17-1A were cloned under the control of P(10) and Polyhedrin promoters in the pFastBac dual vector, respectively. Gene expression cassettes carrying the HC and LC genes were transposed into a bacmid in Escherichia coli (DH10Bac). The transposed bacmid was transfected to Sf9 insect cells to generate baculovirus expressing mAb CO17-1A. Confocal immunofluorescence and Western blot analyses confirmed expression of mAb CO17-1A in baculovirus-infected insect cells. The optimum conditions for mAb expression were evaluated at 24, 48, and 72 h after the virus infection at an optimum virus multiplicity of infection of 1. Expression of mAb CO17-1A in insect cells significantly increased at 72 h after infection. HPLC analysis of glycosylation status revealed that the insect-derived mAb (mAb(I)) CO17-1A had insect specific glycan structures. ELISA showed that the purified mAb(I) from cell culture supernatant specifically bound to SW948 human colorectal cancer cells. Fluorescence-activated cell sorting analysis showed that, although mAb(I) had insect specific glycan structures that differed from their mammalian counterparts, mAb(I) similarly interacted with CD64 (FcgammaRI) and Fc of IgG, compared to the interactions of mammalian-derived mAb. These results suggest that the baculovirus insect cell expression system is able to express, assemble, and secrete biofunctional full size mAb.
Song
Mira
M
Department of Biological Science, College of Natural Sciences, Wonkwang University, Iksan, Jeonbuk 570-749, Korea.
Park
Da-Young
DY
Kim
Youngkwan
Y
Lee
Kyung-Jin
KJ
Lu
Zhe
Z
Ko
Kinarm
K
Choo
Young Kug
YK
Han
Yeon Soo
YS
Ahn
Mi-Hyun
MH
Oh
Doo-Byoung
DB
Ko
Kisung
K
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism* / Q000627:therapeutic use; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000378:metabolism* / Q000627:therapeutic use; D016367:Baculoviridae / Q000502:physiology*; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D011134:Polysaccharides / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D017452:Receptors, IgG; D020298:U937 Cells
D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D011134:Polysaccharides; D017452:Receptors, IgG