BACKGROUND & OBJECTIVE: Idiotypic determinant (Id) of malignant B lymphocyte surface membrane immunoglobulin (SmIg) is not only a specific marker for chronic B-cell lymphatic leukemia (B-CLL) but also a specific antigen as an attractive target for active immunotherapy. However, as a small self antigen, it has poor immunogenicity. As an important molecule for antigen presentation, heat shock protein 70 (Hsp70) can efficiently strengthen the immunogenicity of antigen. In this study, we prepared Hsp70 and idiotypic determinant single-chain antibody (Id-ScFv) fragment against SmIg in patient with B-CLL and explored the in vitro antitumor effect of peptide complex Hsp70-Id and the possible immune mechanism. METHODS: Id-ScFv and human Hsp70 proteins were expressed in E.coli and identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA), and purified by metal chelate chromatography and DEAE ion-exchange chromatography. The purified Hsp70 was combined with the prepared Id-ScFv to produce peptide complex Hsp70-Id in vitro. Peripheral blood mononuclear cells (PBMCs) from healthy human were stimulated with Id-ScFv, HSP70 and HSP70-Id, separately. The proliferation rate of PBMCs was determined by MTT assay; the changes of T lymphocyte subsets were assessed by flow cytometry (FCM)û the levels of interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF-alpha) in supernatant were detected by ELISA. Killing efficiency of activated PBMCs to chronic B-cell leukemia cell line Daudi, chronic myelogenous leukemia cell line K562 and hepatoma carcinoma cell line HepG2 were tested by cell counting. RESULTS: Id-ScFv and Hsp70 were expressed successfully in E.coli. The proliferation rate of PBMCs and secreting levels of IL-12 and TNF-alpha of lymphocytes in Hsp70-Id group were significantly higher than those in Id-ScFv and HSP70 groups (P<0.05), which were significantly higher than that in negative control group (P<0.05). The proportion of CD8+ T cells was increased in Hsp70-Id, Id-ScFv and Hsp70 groups, and the most significant increase was observed in Hsp70-Id group. The activated PBMCs in both Hsp70-Id and Id-ScFv groups had stronger cytotoxic activities to Daudi cells than to K562 or HepG2 cells (P<0.05), and the effect was significantly stronger in Hsp70-Id group than in Id-ScFv group (P<0.001). CONCLUSION: Purified Hsp70 and Id-ScFv proteins in a B-CLL patient were prepared successfully. Hsp70-Id complex could significantly enhance specific antitumor effect of PBMCs in vitro through improving the proliferation of PBMCs, activating specific CD8+ T cells, and inducing secretion of Th1 type cytokines.
BACKGROUND & OBJECTIVE: Idiotypic determinant (Id) of malignant B lymphocyte surface membrane immunoglobulin (SmIg) is not only a specific marker for chronic B-cell lymphatic leukemia (B-CLL) but also a specific antigen as an attractive target for active immunotherapy. However, as a small self antigen, it has poor immunogenicity. As an important molecule for antigen presentation, heat shock protein 70 (Hsp70) can efficiently strengthen the immunogenicity of antigen. In this study, we prepared Hsp70 and idiotypic determinant single-chain antibody (Id-ScFv) fragment against SmIg in patient with B-CLL and explored the in vitro antitumor effect of peptide complex Hsp70-Id and the possible immune mechanism. METHODS: Id-ScFv and humanHsp70 proteins were expressed in E.coli and identified by sodium dodecyl sulfatepolyacrylamide gel electropheresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA), and purified by metal chelate chromatography and DEAE ion-exchange chromatography. The purified Hsp70 was combined with the prepared Id-ScFv to produce peptide complex Hsp70-Id in vitro. Peripheral blood mononuclear cells (PBMCs) from healthy human were stimulated with Id-ScFv, HSP70 and HSP70-Id, separately. The proliferation rate of PBMCs was determined by MTT assay; the changes of T lymphocyte subsets were assessed by flow cytometry (FCM)û the levels of interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF-alpha) in supernatant were detected by ELISA. Killing efficiency of activated PBMCs to chronic B-cell leukemia cell line Daudi, chronic myelogenous leukemia cell line K562 and hepatoma carcinoma cell line HepG2 were tested by cell counting. RESULTS: Id-ScFv and Hsp70 were expressed successfully in E.coli. The proliferation rate of PBMCs and secreting levels of IL-12 and TNF-alpha of lymphocytes in Hsp70-Id group were significantly higher than those in Id-ScFv and HSP70 groups (P<0.05), which were significantly higher than that in negative control group (P<0.05). The proportion of CD8+ T cells was increased in Hsp70-Id, Id-ScFv and Hsp70 groups, and the most significant increase was observed in Hsp70-Id group. The activated PBMCs in both Hsp70-Id and Id-ScFv groups had stronger cytotoxic activities to Daudi cells than to K562 or HepG2 cells (P<0.05), and the effect was significantly stronger in Hsp70-Id group than in Id-ScFv group (P<0.001). CONCLUSION: Purified Hsp70 and Id-ScFv proteins in a B-CLL patient were prepared successfully. Hsp70-Id complex could significantly enhance specific antitumor effect of PBMCs in vitro through improving the proliferation of PBMCs, activating specific CD8+ T cells, and inducing secretion of Th1 type cytokines.