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Production and characterisation of a recombinant scFv reactive with human gastrointestinal carcinomas
1Department of Biochemistry and Molecular Biology, Cancer Research Institute, Seoul National University College of Medicine, Seoul, 110-799, Korea.
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-chain-encoding 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.
Insights
Researchers engineered SC142 single-chain fragments to improve tumor penetration for cancer diagnostics and therapeutics. These fragments maintain the activity of the original SC142 antibody, showing promise for immunodiagnostic and immunotherapeutic applications.
Area of Science:
- Oncology
- Immunology
- Biotechnology
Background:
- SC142-reactive antigen, highly glycosylated glycoproteins, are expressed on gastric and colon cancers but not normal tissues.
- Murine SC142 antibody targets this antigen but has limitations including immunogenicity and poor tumor penetration due to large size.
Purpose of the Study:
- To engineer recombinant SC142 single-chain fragments (SC142 SCFv) with improved tumor penetration potential.
- To evaluate the activity, integrity, and binding characteristics of the novel SC142 SCFv.
Main Methods:
- Production of SC142 SCFv using polymerase chain reaction and E. coli expression systems.
- Confirmation of activity via immunohistochemistry, integrity via SDS-PAGE and Western blotting.
- Assessment of origin and binding kinetics using competitive ELISA and BIAcore biosensor experiments.
Main Results:
- SC142 SCFv demonstrated similar activity to the parent SC142 antibody.
- Integrity and origin of SC142 SCFv were confirmed through molecular and biochemical analyses.
- SC142 SCFv exhibited an ideal dissociation rate constant, suitable for tumor imaging.
Conclusions:
- Engineered SC142 SCFv overcome the limitations of the parent antibody, offering enhanced tumor penetration.
- These novel fragments show significant potential as immunodiagnostic and immunotherapeutic reagents for gastric and colon cancers.