Abstract
BackgroundCEA 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.MethodsThe 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.ResultsThe 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.ConclusionThe 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.
Highlights
carcinoembryonic antigen (CEA) is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy
Murine (CEA)-specific monoclonal antibodies were successfully tested in animals for their capacity to accurately localize tumors formed by human colorectal carcinoma cell lines with various levels of CEA expression and, as a result, their use is foreseen in radioimmunoguided surgery (RIGS) [7]
Selection and specificity of MA39 scFv In order to isolate phage-displayed specific antibodies, an aliquot of the human synthetic ETH-2 library containing approximately 1 × 1012 transducing units (TU) was panned on biotinylated CEA, as described in Materials and Methods
Summary
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. The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important technological tool in generating new monoclonal antibodies for research and clinical applications. The carcinoembryonic antigen (CEA) is an oncofetal glycoprotein, containing 50% carbohydrate with a molecular weight of approximately 200 kDa [1], overexpressed in several tumor types of epithelial origin and known as an important and extensively used clinical tumor marker for colorectal and other carcinomas [2,3,4]. The CEA serum levels can be used to monitor recurrent disease in the post-surgical surveillance of colorectal cancer [5], but not to localize the lesions
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