Bispecific antibodies are recombinant proteins with novel immunological properties and therapeutic potential. Recombinant protein quality and activity of several bispecific antibodies comprising different variable dom...Bispecific antibodies are recombinant proteins with novel immunological properties and therapeutic potential. Recombinant protein quality and activity of several bispecific antibodies comprising different variable domain combinations with respect to the parental monospecific single chain fragments (scFv) were evaluated after expression in bacteria or mammalian cells. The parental scFv proteins humanized anti-NCAM scFv, murine anti-VEGFR-2 scFv, murine and humanized anti-CD3 scFv, respectively, could successfully be expressed in E. coli, whereas the murine anti-NCAM scFv version could not be reliably detected. Bispecific CD3 × VEGFR-2 and CD3 × NCAM anti-bodies were expressed in the bispecific single chain and the single chain diabody format. However, the diabody derived from the murine anti-NCAM scFv could not efficiently be expressed in E. coli or in mammalian cells. Significant binding of the CD3 × NCAM single chain diabody comprising the humanized version of anti-CD3 and humanized version of anti-NCAM was efficient to both antigens. Nevertheless, binding of the bispecific single chain version to the NCAM antigen was inefficient in comparison to CD3 binding. In conclusion, the data could indicate that the result of scFv expression in bacteria may be predictive for the chances of success for functional expression of more complex bispecific derivatives.展开更多
AIM:To create new diabodies with improved binding activity to antigen of the variable light-variable heavy (VH-VL)oriented single-chain Fv dimers genes(scFv). METHODS:The linker between VH and VL genes was shortened t...AIM:To create new diabodies with improved binding activity to antigen of the variable light-variable heavy (VH-VL)oriented single-chain Fv dimers genes(scFv). METHODS:The linker between VH and VL genes was shortened to 3-5 amino acid residues and cloned into the vector pCANTAB5E.The recombinant plasmids were transformed into TG1 cells and sequenced.The positive transformed cells were infected by M13K07 helper phage to form human recombinant phage antibodies.Expressed products were identified by SDS-PAGE,Western blotting,size exclusion gel chromatography(SEC),ELISA and immunohistochemistry. RESULTS:Three scFv(scFv-3,scFv-4,scFv-5)were constructed successfully with binding ability to hepato-cellular carcinoma 3.5-6 fold greater than their parental scFv.The single-chain Fv dimer(scFv-5,termed BDM3) with the best binding ability was successfully expressed in Yeast pichlia,as shown by SDS-PAGE and Western blotting.SEC results suggested the molecular weight of the expressed products was about 61 kDa.Expressed products showed significantly stronger binding to hepatocellular carcinoma cells than scFv,still having 50% binding activity even after 16 h incubation as 37℃.The purified dimers were bound specifically to the tumor antigen of HCC. CONCLUSION:we have generated scFv dimers by shortening a series of linkers to 3-5 amino acid residues in VH-linker-VL orientation,resulting in highly stable and affinity-improved dimeric molecules.These will become an attractive targeting moiety in immunotherapeutic and diagnostic applications for HCC.展开更多
Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody(BsAb).To choose an ideal format of anti-CD3 x anti-transferrin receptor(TfR)bispecific antibodies fo...Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody(BsAb).To choose an ideal format of anti-CD3 x anti-transferrin receptor(TfR)bispecific antibodies for clinical application,we constructed TfR bispecific T-cell engager(BiTE)in two extensively applied formats,including single-chain tandem singlechain variable fragments(scFvs)and double-chain diabodies,and evaluated their functional characterizations in vitro.Results demonstrated that TfR-BiTE in both formats directed potent killing of TfR+HepG2 cells.However,compared to two・chain diabodies,scFvs were more efficient in antigen binding and TfR target killing.Furthermore,different domain orders in scFvs would also be evaluated because single-TfR-CD3-His was preferable to single-CD3-TfR-His in immunotherapeutic strategies.Thus,the single-chain tandem TfR-CD3 format was favored for further investigation in cancer therapy.展开更多
基金financial support of AK by a grant of the Clotten-Stiftung,Freiburg,GermanyPPM was supported by the Deutsche Forschungsgemeinschaft DFG grant SFB599.
文摘Bispecific antibodies are recombinant proteins with novel immunological properties and therapeutic potential. Recombinant protein quality and activity of several bispecific antibodies comprising different variable domain combinations with respect to the parental monospecific single chain fragments (scFv) were evaluated after expression in bacteria or mammalian cells. The parental scFv proteins humanized anti-NCAM scFv, murine anti-VEGFR-2 scFv, murine and humanized anti-CD3 scFv, respectively, could successfully be expressed in E. coli, whereas the murine anti-NCAM scFv version could not be reliably detected. Bispecific CD3 × VEGFR-2 and CD3 × NCAM anti-bodies were expressed in the bispecific single chain and the single chain diabody format. However, the diabody derived from the murine anti-NCAM scFv could not efficiently be expressed in E. coli or in mammalian cells. Significant binding of the CD3 × NCAM single chain diabody comprising the humanized version of anti-CD3 and humanized version of anti-NCAM was efficient to both antigens. Nevertheless, binding of the bispecific single chain version to the NCAM antigen was inefficient in comparison to CD3 binding. In conclusion, the data could indicate that the result of scFv expression in bacteria may be predictive for the chances of success for functional expression of more complex bispecific derivatives.
基金Supported by Guangdong Province Science and Technology Tackling Key Project,No.2006B19901014the New Drug Project of Guangzhou Committee of Science and Technology,No.2004Z3-E4121
文摘AIM:To create new diabodies with improved binding activity to antigen of the variable light-variable heavy (VH-VL)oriented single-chain Fv dimers genes(scFv). METHODS:The linker between VH and VL genes was shortened to 3-5 amino acid residues and cloned into the vector pCANTAB5E.The recombinant plasmids were transformed into TG1 cells and sequenced.The positive transformed cells were infected by M13K07 helper phage to form human recombinant phage antibodies.Expressed products were identified by SDS-PAGE,Western blotting,size exclusion gel chromatography(SEC),ELISA and immunohistochemistry. RESULTS:Three scFv(scFv-3,scFv-4,scFv-5)were constructed successfully with binding ability to hepato-cellular carcinoma 3.5-6 fold greater than their parental scFv.The single-chain Fv dimer(scFv-5,termed BDM3) with the best binding ability was successfully expressed in Yeast pichlia,as shown by SDS-PAGE and Western blotting.SEC results suggested the molecular weight of the expressed products was about 61 kDa.Expressed products showed significantly stronger binding to hepatocellular carcinoma cells than scFv,still having 50% binding activity even after 16 h incubation as 37℃.The purified dimers were bound specifically to the tumor antigen of HCC. CONCLUSION:we have generated scFv dimers by shortening a series of linkers to 3-5 amino acid residues in VH-linker-VL orientation,resulting in highly stable and affinity-improved dimeric molecules.These will become an attractive targeting moiety in immunotherapeutic and diagnostic applications for HCC.
基金National Natural Science Foundation of China(No.31570937 and No.81871391)Natural Science Foundation of Hubei Province of China(No.2017CFB707)+1 种基金the Fundamental Research Funds for the Central Universities of China(No.HUST:2018KFYYXJJ086)Graduates'Innovation Foundation of Huazhong University of Science and Technology(No.5003510001).
文摘Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody(BsAb).To choose an ideal format of anti-CD3 x anti-transferrin receptor(TfR)bispecific antibodies for clinical application,we constructed TfR bispecific T-cell engager(BiTE)in two extensively applied formats,including single-chain tandem singlechain variable fragments(scFvs)and double-chain diabodies,and evaluated their functional characterizations in vitro.Results demonstrated that TfR-BiTE in both formats directed potent killing of TfR+HepG2 cells.However,compared to two・chain diabodies,scFvs were more efficient in antigen binding and TfR target killing.Furthermore,different domain orders in scFvs would also be evaluated because single-TfR-CD3-His was preferable to single-CD3-TfR-His in immunotherapeutic strategies.Thus,the single-chain tandem TfR-CD3 format was favored for further investigation in cancer therapy.
