Optimized selection of anti-tumor recombinant antibodies from phage libraries on intact cells.
Generation of human recombinant antibody libraries displayed on the surface of the filamentous phage and selection of specific antibodies against desirable targets allows production of fully human antibodies usable for repeated administration in humans. Various lymphoid tissues from immunized donors, such as lymph nodes or peripheral blood lymphocytes from individuals with tumor or lymphocytes infiltrating tumor masses may serve as a source of specific anti-tumor antibody repertoire for generation of tumor-focused phage display libraries. In the case of lack of tumor-associated antigens in the purified form, high affinity anti-tumor antibodies can be isolated through library panning on whole cells expressing these antigens. However, affinity selection against cell surface specific antigens within highly heterogeneous population of molecules is not a very efficient process that often results in the selection of unspecific antibodies or antibodies against intracellular antigens that are generally useless for targeted immunotherapy. In this work, we developed a new cell-based antibody selection protocol that, by eliminating the contamination of dead cells from the cell suspension, dramatically improves the selection frequency of anti-tumor antibodies recognizing cell surface antigens.
Biotechnology, Sigma-tau, SpA, via Pontina km 30.400, Pomezia (RM) 00040, Italy. Electronic address: emiliano.pavoni@email.it.
Emiliano
E
Pavoni
Paola
P
Vaccaro
Anna Maria
AM
Anastasi
Olga
O
Minenkova
D000888:Antibodies, Anti-Idiotypic / Q000235:genetics / Q000276:immunology; D000912:Antibodies, Neoplasm / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D000951:Antigens, Neoplasm / Q000276:immunology; D000954:Antigens, Surface / Q000276:immunology; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D061986:MCF-7 Cells; D019151:Peptide Library; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D057127:Single-Chain Antibodies / Q000276:immunology*
D000888:Antibodies, Anti-Idiotypic; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies
BSA; CEA; Cell-based selection; ELISA; FACS; HRP; PBS; PEG; PFU; Phage antibody library; RT; SEREX; TIL; bovine serum albumin; carcino embryonic antigen; enzyme-linked immunosorbent assay; fluorescence-activated cell sorting; horseradish peroxidase; phosphate-buffered saline; plaque-forming unit(s); polyethylene glycol; room temperature; scFv; serological identification of antigens by recombinant expression cloning; single-chain variable antibody fragment; tumor-infiltrating lymphocytes