| There is still no really effective treatment of nasopharyngeal carcinoma (NPC), despite the use of radical surgery, and therefore seems necessary to develop adoptive immunological approaches. According to the immune network hypothesis proposed by Jerne, {3 type Ab2 that acts as an internal image of the antigen should make activation of both humoral and cellular immune responses. Recently, the potential of Ab2(3 as an immunizing agent for cancer patients has been demonstrated in preliminary laboratory and clinical investigations. In this experiment, we construct a phage human anti-idiotypic antibody library with in vitro immunization, EBV transformation and phage antibody library technique. Anti-idiotypic antibodies that could mimic the NPC associated antigen were selected from phage anti-idiotypic antibody library, 5 |3 type phage anti-idiotypic antibodies were isolated, examined and sequenced.Peripheral blood mononuclear cells (PBMCs) isolated from 10 patients with NPC were immunized in vitro with anti-NPC monoclonal antibody (FC2) for 6 to 8 days. The immunized PBMCs were transformed by EBV. Detection of Sandwich ELISA showed that of 10 NPC' patients, 8 patients' B lymphocytes transformed by EBV could produce anti-idiotypic antibodies. RNAs was extracted from ELISA-positive B cells with TRIzol. Heavy chain VH-CH1 and light chain VL-CL cDNAs were amplified by RT-PCR. 5 types of VH and 7 types of VL genes were re-amplified and combined to amplify 14 types of single chain fragments of variable region (scFv) genes through (Gly4Ser) 3 linker with PCR. ScFv genes digested with Sfi I were cloned into fUSE5vectors and transformed into MC1061 with electroporation. Phageanti-idiotypic antibody library was obtained with 1.5 X 108 tetracycline-resistant colonies, in which the percentage of full-length scFv genes inserts was 70%. Then, the library was subjected to four rounds of positive selection with purified FC2. Enrichment of bounded phages was 11210 times. Phages that bound FC2 were recovered. 270 clones that were randomly selected from this selected library were further screened by Sandwich ELISA. 91 phage anti-idiotypic antibodies were picked; the positive ratio was 33.7%. To further screen (3 type anti-idiotypic antibodies, a binding inhibition test was carried out, in which a fixed amount of HRP-conjugated FC2 was reacted with different amounts of phage anti-idiotypic antibodies on ELISA. It was found that 15 of 91 anti-idiotypic antibodies could inhibit the binding of FC2 to NPC cell lines; the inhibition ratio was almost 48.47% ?9.30. Furthermore, the. inhibition in 5 phage anti-idiotypic antibodies was dose-dependent. This indicated that these 5 phage anti-idiotypic antibodies shared the same binding epitope with NPC associated antigen and might be (3 type anti-idiotypic antibodies. These 5 phage anti-idiotypic antibodies were further analyzed by DNA sequencing. The lengths of D83, E92, G22, 150, 154 were 228bp, 720bp, 720bp, 222bp and 222bp, respectively. The VDJ regions of G22, 150, 154 belonged to VH4-39-D4-1 1-JH3-linker-V1-19-JL2, VH4-4-D4-11-JH6 and VH4-31-D4-11-JH6, respectively. The number of their GenBank were AB019439, AB019441 and AB019439, respectively. E92 had the same VDJ regions with G22; D83 had the same VDJ regions with 150.Phage human anti-idiotypic antibody library was constructed from PBMCs of NPC patients with phage antibody library technique in combination with in vitro immunization and EBV transformation technique. The phage anti-idiotypic antibodies were isolated and examined with Sandwich ELISA, binding inhibition assay and DNAsequencing. The isolated phage anti-idiotypic antibodies could inhibit the binding of NPC monoclonal antibody (FC2) to NPC cell lines, which indicated that they shared the same binding epitope with NPC cell and were capable of mimicking the NPC associated antigen. The human anti-idiotypic antibody scFv and CDR3 fragments of NPC might be further developed and applied to clinical therapy.
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