Construction and selection of human anti-idiotypic antibody single chain variable fragments or CDR3 fragments of nasopharyngeal carcinoma.
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). Detection showed that of 10 NPC patients, 8 patients' B cells immunized by FC2 and transformed by EBV produced anti-idiotypic antibodies to NPC. Five types of VH genes and 7 types of VL genes were obtained by RT-PCR amplification and then connected with (Gly4Ser)3 linker to form 14 types of scFv genes. ScFv genes digested with Sfi I were cloned into vector fUSE5 and transformed into E. coli MC 1061. Phage anti-idiotypic antibody library with 1.5 x 10(8) clones was obtained. After four rounds of panning, 270 phage clones were selected randomly and 91 FC2-positive clones were obtained by Sandwich ELISA, the positive ratio was 33.7%. 5 clones (D83, E92, G22, I50, I54), which might display beta type Ab2 scFv, were selected by binding inhibition test. These 5 phage anti-idiotypic antibodies were further analyzed by DNA sequencing. The VDJ regions of G22, I50, I54 belonged to VH4-39-D4-11-JH3-linker-V1-19-JL2, VH4-4-D4-11-JH6 and VH4-31-D4-11-JH6, respectively. E92 had the same VDJ regions with G22; D83 had the same VDJ regions with 150. So, a strategy for preparing and selecting beta type Ab2 scFv or CDR by means of immunization in vitro, EBV transformation and phage display technique is feasible, which paves a way for preparing cancer vaccine using beta type Ab2 scFv.
He
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Cancer Research Institute, Xiang-Ya School of Medicine, Central South University, China.
Li
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Zhu
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D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002277:Carcinoma / Q000276:immunology* / Q000378:metabolism; D002471:Cell Transformation, Neoplastic; D022801:Complementarity Determining Regions / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000378:metabolism; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D007963:Leukocytes, Mononuclear / Q000378:metabolism / Q000821:virology; D051379:Mice; D009303:Nasopharyngeal Neoplasms / Q000276:immunology* / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013997:Time Factors
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D017931:DNA Primers; D007135:Immunoglobulin Variable Region; D019151:Peptide Library