Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein.
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.
Pavoni
Emiliano
E
Kenton Labs, c/o Sigma-Tau, via Pontina, km 30,400, 00040 Pomezia (RM), Italy. pavoni@kenton.it
Flego
Michela
M
Dupuis
Maria Luisa
ML
Barca
Stefano
S
Petronzelli
Fiorella
F
Anastasi
Anna Maria
AM
D'Alessio
Valeria
V
Pelliccia
Angela
A
Vaccaro
Paola
P
Monteriù
Giorgia
G
Ascione
Alessandro
A
De Santis
Rita
R
Felici
Franco
F
Cianfriglia
Maurizio
M
Minenkova
Olga
O
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
D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D002272:Carcinoembryonic Antigen; D000939:Epitopes; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D004247:DNA