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Open Access Highly Accessed Research article

Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein

Emiliano Pavoni1, Michela Flego2, Maria Luisa Dupuis2, Stefano Barca2, Fiorella Petronzelli3, Anna Maria Anastasi3, Valeria D'Alessio3, Angela Pelliccia3, Paola Vaccaro1, Giorgia Monteriù1, Alessandro Ascione2, Rita De Santis3, Franco Felici14, Maurizio Cianfriglia2 and Olga Minenkova1*

Author Affiliations

1 Kenton Labs, c/o Sigma-Tau, via Pontina, km 30.400, 00040 Pomezia (RM), Italy

2 Pharamcogenetics, Drug Resistance & Experimental Therapeutics Section, Department of Drug Research and Evaluation of Italian National Institute of Health (Istituto Superiore di Sanità), Viale R. Elena 299, 00161 Rome, Italy

3 Immunology Department, Sigma-Tau, via Pontina, km 30.400, 00040 Pomezia (RM), Italy

4 Department of Microbiology, Genetics, Molecular Biology, University of Messina, Messina, 98100, Italy

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BMC Cancer 2006, 6:41  doi:10.1186/1471-2407-6-41

Published: 24 February 2006

Abstract

Background

CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration.

Methods

The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized.

Results

The scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells.

Conclusion

The binding affinity of the scFv E8 is in a range for efficient, in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance.