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.
[Show abstract] [Hide abstract]
ABSTRACT: IgG antibodies have evolved to be flexible to bind to epitopes located over a wide spatial range. The two Fabs in an IgG antibody are linked together as if each Fab is at the end of a linear, flexible molecule. PEG was used as a scaffold molecule to link two Fabs together to give Fab-PEG-Fab molecules, or FpFs. Preparation of FpFs was achieved with reagents that undergo site-specific conjugation at each PEG terminus by bis-alkylation with the two cysteine thiols from a disulfide bond. This allowed each Fab to be conjugated to the PEG scaffold in essentially the same region that each Fab is linked in an IgG. Fabs were sourced directly (e.g. ranibizumab) or monoclonal IgG antibodies were proteolytically digested to obtain the Fabs. This allowed the resulting FpFs to be directly compared to parent IgGs. PEG scaffolds of 6, 10 and 20 kDa were used to make the corresponding FpFs. Dynamic light scatting data suggested the resulting FpFs were similar in size to an IgG antibody and about half the size of a 20 kDa PEGylated-Fab. The solution size of PEG conjugated proteins is known to be dominated by the extended solution structure of PEG, so it is thought the smaller size of the FpFs is due to interactions between the two Fabs. Anti-VEGF and anti-Her2 FpFs were prepared and evaluated. The FpFs displayed similar apparent affinities to their parent IgGs. Slower dissociation rates were observed for the anti-VEGF FpFs compared to bevacizumab. The anti-VEGF FpFs also displayed in vitro anti-angiogenic properties comparable or better than bevacizumab. These first studies indicate that FpFs warrant further examination in a therapeutic indication where the presence of the Fc may not be required.Bioconjugate Chemistry 09/2013; 24(11). DOI:10.1021/bc400246z · 4.82 Impact Factor
[Show abstract] [Hide abstract]
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. DOI:10.1007/s11427-007-0058-5 · 1.61 Impact Factor