Isolation and characterization of antibodies against three consecutive Tn-antigen clusters from a phage library displaying human single-chain variable fragments

Abstract

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.

Recognition of the Thomsen-Friedenreich Pancarcinoma Carbohydrate Antigen by a Lamprey Variable Lymphocyte Receptor

“…Most often, monoclonal antibodies are isolated from mice immunized with the glycan of interest, but this process is laborious, and most human glycans are not particularly immunogenic in mice, probably due to their conserved structures in these species, which gives rise to self-tolerance. Other strategies include directed evolution of antibodies (7) or lectins (3,8), carbohydrate binding peptides (9), and glycan-specific aptamers (10), but none has proven generally useful.…”

Screening for a Single-Chain Variable-Fragment Antibody That Can Effectively Neutralize the Cytotoxicity of the Vibrio parahaemolyticus Thermolabile Hemolysin

“…It has also been used for selective molecular targeting in cancer research for conditions such as lymphatic invasion vessels, colon cancer, and hepatocarcinoma (27,29,33,43). Furthermore, scFv antibody generation has been used extensively to generate ligands for detecting pathogenic germs in vitro and in vivo (8,22,38,39).…”

Purification and refolding of anti-T-antigen single chain antibodies (scFvs) expressed in Escherichia coli as inclusion bodies

“…Inclusion bodies isolated from E. coli cells were denatured in 3.5 M Gdn-HCl. Solubilized His-tagged scFv proteins were purified using Ni anti-Tn-antigen, and anti-T-antigen scFv proteins have been isolated from a phage library displaying human scFvs that had been characterized after purification of scFv proteins expressed in different expression systems (8)(9)(10)(11)(12). Escherichia coli expression systems are the most prevalent systems for production of recombinant proteins (13,14).…”

“…The current authors and their colleagues previously isolated genes encoding human scFv proteins with specificity against various carbohydrate moieties from a phage library displaying human scFv proteins (8)(9)(10)(11)(12). Eleven anti-T-antigen phage clones were isolated from a phage library and categorized into four groups (11).…”