Generation and antitumor effects of an engineered and energized fusion protein VL-LDP-AE composed of single-domain antibody and lidamycin

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.

Antitumor efficacy of the scFv-based fusion protein and its enediyne-energized analogue directed against epidermal growth factor receptor

“…The PCR profile was as follows: 30 cycles of 1 min degeneration at 941C, 2 min annealing at 581C, 2 min extension at 721C, followed by a final extension for 10 min at 721C. After purification, scfv PCR product and the vector pET30(a)-ldp containing the LDP-encoded fragment [13] were digested with Nde I and EcoR I restriction enzymes (Takara, Dalian, China), and were then ligated and used to transform competent Escherichia coli (E. coli) strain DH5a (Novagen, Madison, Wisconsin, USA). Transformed cells were plated onto Luria-Bertani agar plates containing 50 mg/ml of kanamycin, and then the plates were inverted and incubated at 371C overnight.…”

Small antibody fusion proteins with complementarity-determining regions and lidamycin for tumor targeting therapy

“…The detailed procedure was described previously (14), and the final affintiy constant was determined by Graphpad Prism 5 software (San Diego, CA, USA).…”

“…Human Bel-7402 and HepG2 hepatoma cell lines were seeded in 96-well plate at a density of 1×10 4 cells/well and cultivated overnight at 37°C. The following procedure was performed according to that of our previous study (14). …”

The novel structure make LDM effectively remove CD123+ AML stem cells in combination with interleukin 3

“…As one of the most potent antitumoral agents ever discovered, LDM is currently undergoing phase II clinical trial in China. 11 After combined with anti-CD20Fab 12 or anti-type IV collagenase monoclonal antibody, 13 AE exhibited marked inhibition on a panel of transplantable tumors in mice, which include leukemia L1210, P388, ascites hepatoma H22, sarcoma 180 and melanoma Harding-Passey. Also, and importantly, LDM consist of 2 independent parts: an apoprotein moiety(LDP) and a non-protein chromophore extractable (AE) which could be reassembled and depart freely.…”