Effects of interlinker sequences on the biological properties of bispecific single-chain antibodies
ABSTRACT Single-chain bispecific antibody (scBsAb) is one of the promising genetic engineering antibody formats for clinical application.
But the effects of interlinker sequences on the biological properties of bispecific single-chain antibodies have not been
studied in detail. Three interlinker sequences were designed and synthesized, and denominated as Fc, HSA, 205C′, respectively.
Universal vectors with these different interlinker sequences for scBsAb expression in E. coli were constructed. A model scBsAb based on a reshaped single-chain antibody (scFv) against human CD3 and a scFv directed against
human ovarian carcinoma were generated and expressed in E. coli. The results of SDS-PAGE and Western blot showed that the different interlinker sequences did not affect the expression level
of scBsAb. However, as demonstrated by ELISA and pharmacokinetics studies performed in mice, scBsAbs with different interlinker
sequences had difference in the antigen-binding activities and terminal half-life time (T
1÷2β) in vivo, the interlinker HSA could remarkably prolong the retention time of scBsAb in blood. These results indicated that the peptide
sequence of interlinker could affect important biological properties of scBsAb, such as antigen-binding properties and stability
in vivo. So, selection of an appropriate interlinker sequence is very important for scBsAb construction. Optimal interlinker can
bring scBsAb biological properties more suitable for clinical application.
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ABSTRACT: 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 compared 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 IC(50) values of 8.55 x 10(-12) and 1.70 x 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.Science in China Series C Life Sciences 09/2007; 50(4):447-56. · 1.61 Impact Factor