title: Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Kenton Labs, c/o Sigma-Tau, via Pontina, km 30,400, 00040 Pomezia (RM), Italy. pavoni@kenton.it
authors.forename: Emiliano
authors.initials: E
authors.lastname: Pavoni
authors.forename: Michela
authors.initials: M
authors.lastname: Flego
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Dupuis
authors.forename: Stefano
authors.initials: S
authors.lastname: Barca
authors.forename: Fiorella
authors.initials: F
authors.lastname: Petronzelli
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Anastasi
authors.forename: Valeria
authors.initials: V
authors.lastname: D'Alessio
authors.forename: Angela
authors.initials: A
authors.lastname: Pelliccia
authors.forename: Paola
authors.initials: P
authors.lastname: Vaccaro
authors.forename: Giorgia
authors.initials: G
authors.lastname: Monteriù
authors.forename: Alessandro
authors.initials: A
authors.lastname: Ascione
authors.forename: Rita
authors.initials: R
authors.lastname: De Santis
authors.forename: Franco
authors.initials: F
authors.lastname: Felici
authors.forename: Maurizio
authors.initials: M
authors.lastname: Cianfriglia
authors.forename: Olga
authors.initials: O
authors.lastname: Minenkova
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000918:Antibody Specificity; D000970:Antineoplastic Agents / Q000494:pharmacology; D001419:Bacteria / Q000378:metabolism; D019921:Biotinylation; D015153:Blotting, Western; D002272:Carcinoembryonic Antigen / Q000737:chemistry*; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry; D005434:Flow Cytometry; D015723:Gene Library; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D007150:Immunohistochemistry; D007700:Kinetics; D008175:Lung Neoplasms / Q000378:metabolism; D008545:Melanoma / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009362:Neoplasm Metastasis; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D002272:Carcinoembryonic Antigen; D000939:Epitopes; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D004247:DNA