Type IV collagenase plays a pivotal role in invasion,metastasis and angiogenesis of tumor.Single domain antibodies are attractive as tumor-targeting vehicle because of their much smaller size com-pared with antibody m...Type IV collagenase plays a pivotal role in invasion,metastasis and angiogenesis of tumor.Single domain antibodies are attractive as tumor-targeting vehicle because of their much smaller size com-pared with antibody molecules produced by conventional methods.Lidamycin(LDM)is a potent enediyne-containing antitumor antibiotic.In this study an engineered and energized fusion protein VL-LDP-AE composed of lidamycin and VL domain of mAb 3G11 directed against type IV collagenase was prepared using a novel two-step method.First a VL-LDP fusion protein was constructed by DNA recombination.Secondly VL-LDP-AE was obtained by molecular reconstitution.In MTT assay,VL-LDP-AE showed potent cytotoxicity to HT-1080 cells and KB cells with IC50 values of 8.55×10-12 and 1.70×10-11 mol/L,respectively.VL-LDP-AE showed antiangiogenic activity in chick chrorioallantoic membrane(CAM)assay and tube formation assay.In in vivo experiments,VL-LDP-AE was proved to be more effective than free LDM against the growth of subcutaneously transplanted hepatoma 22 in mice.Drugs were given intravenously on day 3 and 10 after tumor transplantation.Compared in terms of maximal tolerated doses,VL-LDP-AE at 0.25 mg/kg suppressed the tumor growth by 89.5%,LDM at 0.05 mg/kg by 69.9%,and mitomycin at 1 mg/kg by 35%.Having a molecular weight of 25.2 kDa,VL-LDP-AE was much smaller than other reported antibody-based drugs.The results suggested that VL-LDP-AE would be a promising candidate for tumor targeting therapy.And the 2-step approach could serve as a new technology platform for making a series of highly potent engineered antibody-based drugs for a variety of cancers.展开更多
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 Lymph...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.展开更多
基金Supported by the National High Technology Research and Development Program of China(863 Program)(Grant No.2006AA02A255)
文摘Type IV collagenase plays a pivotal role in invasion,metastasis and angiogenesis of tumor.Single domain antibodies are attractive as tumor-targeting vehicle because of their much smaller size com-pared with antibody molecules produced by conventional methods.Lidamycin(LDM)is a potent enediyne-containing antitumor antibiotic.In this study an engineered and energized fusion protein VL-LDP-AE composed of lidamycin and VL domain of mAb 3G11 directed against type IV collagenase was prepared using a novel two-step method.First a VL-LDP fusion protein was constructed by DNA recombination.Secondly VL-LDP-AE was obtained by molecular reconstitution.In MTT assay,VL-LDP-AE showed potent cytotoxicity to HT-1080 cells and KB cells with IC50 values of 8.55×10-12 and 1.70×10-11 mol/L,respectively.VL-LDP-AE showed antiangiogenic activity in chick chrorioallantoic membrane(CAM)assay and tube formation assay.In in vivo experiments,VL-LDP-AE was proved to be more effective than free LDM against the growth of subcutaneously transplanted hepatoma 22 in mice.Drugs were given intravenously on day 3 and 10 after tumor transplantation.Compared in terms of maximal tolerated doses,VL-LDP-AE at 0.25 mg/kg suppressed the tumor growth by 89.5%,LDM at 0.05 mg/kg by 69.9%,and mitomycin at 1 mg/kg by 35%.Having a molecular weight of 25.2 kDa,VL-LDP-AE was much smaller than other reported antibody-based drugs.The results suggested that VL-LDP-AE would be a promising candidate for tumor targeting therapy.And the 2-step approach could serve as a new technology platform for making a series of highly potent engineered antibody-based drugs for a variety of cancers.
基金supported by the National High Technology Research and Development Program of China(Grant No. 2006AA02A255)the National Natural Science Foundation of China(Grant No. 30701029)the National Science and Technology Major Projects(Grant Nos.2009ZX09103-720 and 2010ZX09401-407)
文摘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.
