Isolation and characterization of antibodies against three consecutive Tn-antigen clusters from a phage library displaying human single-chain variable fragments.
The Tn-antigen, GalNAcalpha-Ser/Thr, is a tumour-associated carbohydrate antigen that may provide a sensitive and specific marker for pre-clinical detection of carcinoma and a target for cancer therapies. We recently reported that MLS128 monoclonal antibody treatment significantly inhibited colon and breast cancer cell growth. On the basis of our observations, the present study aimed to produce human anti-Tn-antigen antibodies with specificity similar to that of MLS128 monoclonal antibody, which recognizes a structure of three consecutive Tn-antigens (Tn3). Six phage clones displaying human single-chain variable fragments (scFvs) were isolated from a newly constructed phage library by panning and screening with a synthetic Tn3-peptide. Deduced amino-acid sequences of six anti-Tn3 scFvs exhibited a high degree of homology. Of those, anti-Tn3 4E10 and 4G2 scFv proteins were successfully purified from phage-infected Escherichia coli to near homogeneity. Surface plasmon resonance analyses revealed a K(D) of purified scFv proteins for Tn3 on an order of 10(-7) M, which is high for carbohydrate-specific monovalent antibodies. Further analyses suggested that both scFv proteins also bind to Tn2 and cultured colon and breast cancer cells. These results demonstrated the potential for use of these scFvs in developing antibody therapeutics targeting colon and breast cancer.
Sakai
Keiko
K
Department of Applied Biochemistry, Tokai University School of Engineering, Kanagawa 259-1292, Japan.
Yuasa
Noriyuki
N
Tsukamoto
Kazuhiro
K
Takasaki-Matsumoto
Ayano
A
Yajima
Yukiko
Y
Sato
Reiko
R
Kawakami
Hiroko
H
Mizuno
Mamoru
M
Takayanagi
Atsushi
A
Shimizu
Nobuyoshi
N
Nakata
Munehiro
M
Fujita-Yamaguchi
Yoko
Y
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification / Q000627:therapeutic use; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000276:immunology*; D001435:Bacteriophages; D001943:Breast Neoplasms / Q000276:immunology / Q000628:therapy; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000276:immunology / Q000628:therapy; D005455:Fluorescent Antibody Technique; D006020:Glycopeptides / Q000138:chemical synthesis / Q000276:immunology*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology; D019151:Peptide Library; D017386:Sequence Homology, Amino Acid; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000302:isolation & purification* / Q000627:therapeutic use; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D015295:Antigens, Tumor-Associated, Carbohydrate; D006020:Glycopeptides; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D057127:Single-Chain Antibodies; C040286:Tn antigen