Isolation and characterization of anti-T-antigen single chain antibodies from a phage library.
T-antigen (Galbeta1-3GalNAc-Thr/Ser) also known as Thomsen-Friedenreich (TF) antigen is the core 1 structure of O-linked mucin type glycans. In normal epithelium, the disaccharide structure is masked by terminal carbohydrate moieties, but is uncovered in most primary and metastatic epithelial malignant tumors. For the purpose of establishing cancer diagnosis and therapeutics, anti-T-antigen antibodies were isolated from a phage library displaying human single chain antibodies. A strategy similar to the previously published method (Sakai et al. Biochemistry. 2007; 46:253-262) was used to screen T-antigen specific antibodies, except that a different type of glycolipid was used for panning and screening. Eleven phage clones were isolated and characterized by DNA sequencing and ELISA, which revealed 4 groups of clones with T-antigen binding activity. One single chain antibody (scFv) protein, derived from phage clone 1G11, was expressed in Escherichia coli and purified to near homogeneity by two column chromatographies. ELISA and surface plasmon resonance analyses revealed that the purified 1G11 scFv bound to the T-antigen moiety of the neoglycolipid used. This study not only demonstrated the validity of our previously introduced strategy employing the phage display technology in constructing human scFvs against various carbohydrate antigens, but also provided us with various scFv genes that can lead to future development of antibody-based therapeutics.
Matsumoto-Takasaki
Ayano
A
Department of Applied Biochemistry, Tokai University School of Engineering, Hiratsuka, Kanagawa, Japan.
Horie
Jinichiro
J
Sakai
Keiko
K
Furui
Yoshihiro
Y
Sato
Reiko
R
Kawakami
Hiroko
H
Toma
Kazunori
K
Takayanagi
Atsushi
A
Shimizu
Nobuyoshi
N
Fujita-Yamaguchi
Yoko
Y
D000906:Antibodies / Q000302:isolation & purification*; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000276:immunology*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D019151:Peptide Library; D017422:Sequence Analysis, DNA; D020349:Surface Plasmon Resonance
D000906:Antibodies; D015295:Antigens, Tumor-Associated, Carbohydrate; D017931:DNA Primers; D019151:Peptide Library; C016467:Thomsen-Friedenreich antigen; C025991:Thomsen-Friedenreich antibodies