[Construction and screening of human anti-idiotypic single chain antibodies of nasopharyngeal carcinoma].Ai Zheng. 2004 Feb; 23(2):124-9.AZ
BACKGROUND & OBJECTIVE
The potential of anti-idiotypic antibody as a surrogate of tumor antigen for cancer therapy has been demonstrated in clinical investigations. But at present, many anti-idiotypic antibodies are mouse-original antibodies, which can cause human anti-mouse antibody (HAMA) response and decrease the curative effect. The objective of this study was to construct phage human anti-idiotypic antibody library and select beta type anti-idiotypic single chain antibodies bearing the internal image of the nasopharyngeal carcinoma (NPC) associated antigen to overcome human anti- mouse antibody response caused by application of mouse-original anti-idiotypic antibody.
Peripheral blood mononuclear cells (PBMCs) of patients with NPC were immunized in vitro by anti-NPC monoclonal antibody FC2 and transformed by Epstein-Barr virus (EBV). V(H) and V(L) genes were amplified by RT-PCR and combined to single chain fragments of variable region (scFv) genes. ScFv genes were cloned into vector fUSE5 and transformed into E.coli MC1061 to construct the scFv-displaying phage library. After four rounds of panning with monoclonal antibody (mAb) FC2,the beta type Ab2 scFv were selected by Sandwich ELISA and binding inhibition test.
Of 10 NPC patients, 8 patients showed their B cells immunized by FC2 and transformed by EBV produced anti-idiotypic antibodies to NPC. Five types of VH genes and 7 types of V(L) genes were obtained by RT-PCR amplification and then connected to form 14 scFv genes. ScFv genes were transducted into E.coli MC1061. The library capacity was 1.5x10(8) clones. After panning, 270 phage clones were selected randomly and 91 FC2-positive clones were obtained by Sandwich ELISA, the positive ratio was 33.7%. Five clones,which might display beta type Ab2 scFv, were selected by binding inhibition test.
The strategy for preparing phage anti-idiotypic antibody library and selecting beta type Ab2 scFv by immunization in vitro, EBV transformation, and phage display technique is feasible, which provide a way for preparing cancer vaccine using beta type Ab2 scFv.