Antibody-based fusion proteins are the next generation of antibody therapies for cancer and other diseases.CD20 antigen,which is overexpressed on cell membranes in nearly 95% of cases of B-cell Non-Hodgkin's Lymphoma,is an attractive target for the therapy of B-lymphoid malignancies.Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic that now has entered phase II clinical trials.In this study,we prepared an engineered fusion protein,scFv-LDP,consisting of an anti-CD20 scFv fragment and the apoprotein LDP of LDM using DNA recombination.After purification and refolding,scFv-LDP was found to bind specifically to CD20-positive lymphoma cells using ELISA and indirect immunofluorescent cytochemical staining assays.The energized fusion protein scFv-LDP-AE was obtained using molecular reconstitution of the active chromophore AE of LDM and scFv-LDP.MTT assay revealed potent cytotoxicity of scFv-LDP-AE to CD20-positive Raji and Daudi cells,with IC 50 values of 1.21×10-11 and 6.24×10-11 mol L-1,respectively.An in vivo subcutaneous xenograft model of CD20-positive B cell lymphoma in BALB/c (nu/nu) mice was also utilized.Drugs were given intravenously on day 14 and 21 after tumor transplantation.In terms of maximal tolerated doses,scFv-LDP-AE at 0.3 mg kg-1 suppressed tumor growth by 79.3%,and LDM at 0.05 mg kg-1 by 68.6% (P<0.05).Results suggested scFv-LDP-AE could be a potential candidate for tumor-targeting therapy.
OBJECTIVE To study the cytotoxicity of Lidamycin (LDM)and its induction of apoptosis in Raji and Daudi cells of B-celllymphoma, and the inhibition of growth of the lymphoma Rajixenograft in nude mice.METHODS MTT assay was used to observe the inhibition byLDM on the proliferation of the Raji and Daudi cells. AnnexinV-FITC/PI double-stain, in combination with flow cytometry(FCM), was used to determine the induction of apoptosis by LDMin Raji cells. The B-cell lymphoma Raji xenograft model in nudemice was set up to detect the in vivo antitumor activity of LDM.RESULTS LDM markedly inhibited the proliferation of theRaji and Daudi cells in vitro, with IC_(50) values of 7.13 × 10^(-11) mol/Land 2.91 × 10^(-10) mol/L, respectively. The apoptotic rates of Rajicells were respectively 77.98% and 67.63% at 0.5 nmol/L and 0.25nmol/L of LDM, indicating an obvious induction of apoptosis inRaji cells. LDM inhibited the formation and growth of humanB-cell lymphoma Raji xenograft in nude mice. The inhibitionrates of tumor growth were respectively 74.9% and 65.2% in LDMat dosage group of 0.05 mg/kg and 0.025 mg/kg, suggesting anapparent prolongation of survival time in the nude mouse bearinglymphoma.CONCLUSION LDM can effectively induce apoptosis of theB-cell lymphoma cells and inhibit the xenograft growth in nudemice.
Hong FangShenghua ZhangQingfang MiaoDongsheng XiongYongsu Zhen
