Production and characterisation of a recombinant scFv reactive with human gastrointestinal carcinomas

Abstract

SC142-reactive antigen are highly glycosylated glycoproteins expressed on tissues of gastric and colon cancers but not on normal tissues. Murine SC142 antibody specific for the SC142-reactive antigen has been produced by immunisation with SNU16 stomach cancer cells. However, SC142 antibody has several potential problems such as high immunogenicity and poor tumour penetration owing to their large size. To improve tumour penetration potential in vivo, recombinant single-chain fragments have been produced using the original hybridoma cells as a source of variable heavy-and variable light-chainencoding antibody genes. The use of the polymerase chain reaction, expression cloning technology and gene expression systems in E. coli has led to the production of SC142 single-chain fragments, which was similar in activity to the SC142 parent antibody confirmed by immunohistochemistry. Analysis by DNA sequencing, SDS -PAGE and Western blotting has demonstrated the integrity of the single-chain fragments. Competitive ELISA showed that SC142 single-chain fragments originated from parent SC142 antibody. BIAcore biosensor binding experiments showed that the SC142 single-chain fragments had an ideal dissociation rate constant as a tumour imaging reagent. These results illustrate the potential application of these novel products as an immunodiagnostic and further immunotherapeutic reagent. British Journal of Cancer (2002) Mucins are high-molecular-weight glycoproteins composed by a carbohydrate moiety (mainly O-glycans) that represents 50 -80% of their total mass and peptide core; also called apomucin, it is rich in Thr and Ser (Verma and Davidson, 1994). A variety of alterations of mucins have been described in metaplastic and malignant disease of the stomach (Correa, 1988). To detect those cancer associated alterations of gastric mucin, a number of monoclonal antibodies (mAbs) were developed. Although their specificity for cancer was limited compared with that of genetic markers such as p53, APC, c-met, k-sam and c-erbB-2, immunohistological detection of antigen is much easier than molecular biological analysis of those gene abnormalities, and this is of practical importance.In the previous study, we developed monoclonal antibodies elicited to the human stomach carcinoma cell line SNU16 to detect useful markers for gastric cancer. One of these monoclonal antibodies, SC142, detects an antigen present on adenocarcinoma cells of stomach. Preliminary studies on the molecular properties of the SC142-reactive antigen suggest that the epitope is sensitive to Oglycanase and is expressed on a large, heavily glycosylated molecule indicating that the antigen is probably mucin (Hong et al, 2001).In immunohistochemical studies, SC142-reactive antigen was not detected in normal gastrointestinal epithelium, whereas it was highly expressed in 78% of gastric cancers (29 out of 37) and 87% of colon cancers (27 out of 31) indicating that SC142 antibody can be a valuable tool to detect gastrointestinal cancers.However, the use of whole antibody fo...

Potent inhibition of OKT3-induced T cell proliferation and suppression of CD147 cell surface expression in HeLa cells by scFv-M6-1B9

“…We feel that this result corresponds with the anti-CD3 scFv-B7.1 fusion protein expressed on the surface of HeLa cells binding to T lymphocytes and strongly inducing T-cell activation (Yang et al, 2007b). Furthermore, many reports have described the same binding specificity and affinity of scFv as the monomeric form of their parental antibody (Bedzyk et al, 1990;Pantoliano et al, 1991;Kim et al, 2002). Our results indicate that in the absence of cross-linking and covalent dimerization of parental CD147 mAb, the monomeric form of anti-CD147 molecules can mediate signal transduction and exhibit biological activity in T cells through their antigenspecific domain.…”

Promoting α‐secretase cleavage of beta‐amyloid with engineered proteolytic antibody fragments

“…43 Because of their small size (1/6th the size of intact IgG), low kidney uptake, and rapid blood clearance, scFvs are being increasingly used in cancer research as carrier of radionuclei and drugs to tumors. 44 Therefore, an scFv that specifically hydrolyzes Ab represents a promising therapeutic option for treating AD. We previously identified a light chain antibody mk18, and the scFv derivative c23.5, both of which have a-secretase-like activity.…”

Production of an anti-idiotypic antibody single chain variable fragment vaccine against <italic>Edwardsiella tarda</italic>

“…The scFv is the smallest functional modules of antibody molecule; the lack of Fc domains makes it less immunogenic responsive [36,37], which may result in efficient protection against infection with different serotypes of E. tarda. Additionally, studies demonstrated that when the linker length was ,25 amino acids, scFvs in the V Llinker-V H format had stronger binding activity than those with the V H -linker-V L format [43][44][45].…”

“…In addition, the lack of Fc domain makes scFv less immunogenic responsive. These characteristics make scFv potentially useful in diagnosis and therapy [36,37]. The common flexible linker (Gly 4 Ser) 3 , which connects V H and V L , may substitute for constant region contacts in the Fab and thereby help recover the native binding properties in the scFv [38].…”