The variable domain of heavy chain (VH) and light chain (VL) genes of anti-CD20 monoclonal antibody HL47 were cloned from anti-CD20 ScFv expression vector pCANBTEcd20 by PCR and ligated into vector pYZF to construct c...The variable domain of heavy chain (VH) and light chain (VL) genes of anti-CD20 monoclonal antibody HL47 were cloned from anti-CD20 ScFv expression vector pCANBTEcd20 by PCR and ligated into vector pYZF to construct chimeric anti-CD20 Fab’ fragment expression vector pTZFcd20. Chimeric anti-CD20 Fab’ fragment was expressed in E. coli 16C9 and purified by protein G affinity chromatography. Competitive inhibition assay showed that anti-CD20 Fab’ fragment inhibited binding of HI47 to CD20 on the surface of Daudi cells. Results from MTT assay indicated that chimeric anti-CD20 Fab’fragment inhibited the proliferation of Daudi cells, IC50=69 ug/mL. Affinity of chimeric anti-CD20 Fab’ fragment was determined, Ka was about 8.9×108 (mol/L)-1.展开更多
The increased incidence ofNHL （non-Hodgkin＇s lymphoma）, along with its high mortality rate and pronounced resistance to therapy pose an enormous challenge. Both traditional therapeutic strategies and recently devel...The increased incidence ofNHL （non-Hodgkin＇s lymphoma）, along with its high mortality rate and pronounced resistance to therapy pose an enormous challenge. Both traditional therapeutic strategies and recently developed therapeutic strategies against NHL such as chemoimmunotherapy and targeted therapy have drawbacks. Therefore, novel therapeutic approaches for NHL are urgently needed. Maytansine-loaded PLA-TPGS （polyethylene glycol 1000 succinate-polylactide） nanoparticles were synthesized. And then, rituximab targeting NHL was conjugated together by using EDC （1-ethyl-3-（3-dimethylaminopropyl） carbodiimide） as a coupling agent. The in vitro/vivo antitumor activity was evaluated by Raji cell proliferation inhibition and nude mice xenograft tumor models for NHL. Both the rituximab-conjugated and maytansine-loaded PLA-TPGS nanoparticles （maytansine-NPs （Nanoparticles）-rituximab） and maytansine-loaded PLA-TPGS nanoparticles （maytansine-NPs） presented significant inhibition effect on Raji cell proliferation in a concentration-dependent manner. Compared with conventional maytansine and maytansine-NPs, maytansine-NPs-rituximab showed significantly enhanced cytotoxicity and increased cell apoptosis in Raji cells. The maytansine-NPs-rituximab described in this paper might be a potential formulation for targeting chemotherapy and immunotherapy to CD20＋ B cell malignancies.展开更多
基金This work was supported by the State "863" High-Tech Project (Grant No. 102-09-03-03).
文摘The variable domain of heavy chain (VH) and light chain (VL) genes of anti-CD20 monoclonal antibody HL47 were cloned from anti-CD20 ScFv expression vector pCANBTEcd20 by PCR and ligated into vector pYZF to construct chimeric anti-CD20 Fab’ fragment expression vector pTZFcd20. Chimeric anti-CD20 Fab’ fragment was expressed in E. coli 16C9 and purified by protein G affinity chromatography. Competitive inhibition assay showed that anti-CD20 Fab’ fragment inhibited binding of HI47 to CD20 on the surface of Daudi cells. Results from MTT assay indicated that chimeric anti-CD20 Fab’fragment inhibited the proliferation of Daudi cells, IC50=69 ug/mL. Affinity of chimeric anti-CD20 Fab’ fragment was determined, Ka was about 8.9×108 (mol/L)-1.
文摘The increased incidence ofNHL （non-Hodgkin＇s lymphoma）, along with its high mortality rate and pronounced resistance to therapy pose an enormous challenge. Both traditional therapeutic strategies and recently developed therapeutic strategies against NHL such as chemoimmunotherapy and targeted therapy have drawbacks. Therefore, novel therapeutic approaches for NHL are urgently needed. Maytansine-loaded PLA-TPGS （polyethylene glycol 1000 succinate-polylactide） nanoparticles were synthesized. And then, rituximab targeting NHL was conjugated together by using EDC （1-ethyl-3-（3-dimethylaminopropyl） carbodiimide） as a coupling agent. The in vitro/vivo antitumor activity was evaluated by Raji cell proliferation inhibition and nude mice xenograft tumor models for NHL. Both the rituximab-conjugated and maytansine-loaded PLA-TPGS nanoparticles （maytansine-NPs （Nanoparticles）-rituximab） and maytansine-loaded PLA-TPGS nanoparticles （maytansine-NPs） presented significant inhibition effect on Raji cell proliferation in a concentration-dependent manner. Compared with conventional maytansine and maytansine-NPs, maytansine-NPs-rituximab showed significantly enhanced cytotoxicity and increased cell apoptosis in Raji cells. The maytansine-NPs-rituximab described in this paper might be a potential formulation for targeting chemotherapy and immunotherapy to CD20＋ B cell malignancies.
