A conjugate of an anti-midkine single-chain variable fragment to doxorubicin inhibits tumor growth

Doxorubicin (DOX) was conjugated to a single-chain variable fragment (scFv) against human midkine (MK), and the conjugate (scFv-DOX) was used to target the chemotherapeutic agent to a mouse solid tumor model in which the tumor cells expressed high levels of human MK. The His-tagged recombinant scFv was expressed in bacteria, purified by metal affinity chromatography, and then conjugated to DOX using oxidative dextran (Dex) as a linker. The molecular formula of this immunoconjugate was scFv(Dex)1.3(DOX)20. In vitro apoptosis assays showed that the scFv-DOX conjugate was more cytotoxic against MK-transfected human adenocarcinoma cells (BGC823-MK) than untransfected cells (55.3 ± 2.4 vs 22.4 ± 3.8 percent) for three independent experiments. Nude mice bearing BGC823-MK solid tumors received scFv-DOX or equivalent doses of scFv + DOX for 2 weeks and tumor growth was more effectively inhibited by the scFv-DOX conjugate than by scFv + DOX (51.83 percent inhibition vs 40.81 percent). Histological analysis of the tumor tissues revealed that the highest levels of DOX accumulated in tumors from mice treated with scFv-DOX and this resulted in more extensive tumor cell death than in animals treated with the equivalent dose of scFv + DOX. These results show that the scFv-DOX conjugate effectively inhibited tumor growth in vivo and suggest that antigen-specific scFv may be competent drug-carriers.

Expression and identification of the soluble monoclonal antibody ScFv fragment / 第三军医大学学报

Objective To express soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal carcinomas in E. coli HB2151 and to purify the soluble ScFv and identify its antigen binding activities to find new target vectors for the diagnosis and therapy of colorectal carcinomas. Methods The phage clones displaying ScFv fragment of the monoclonal antibody MC3 were used to infect E. coli HB2151 to express soluble antibodies. The soluble ScFvs were identified by Dot blot and Western blot and their antigen binding activities were determined by ELISA. The VH and VL DNAs of the ScFv DNA derived were sequenced based on the dideoxy method. Results The soluble MC3 ScFvs were expressed successfully. The expression products with a proximate MW of 32?10 3 were mainly secreted into the periplasm. The soluble ScFv containing periplasmatic extracts derived from three clones could inhibit the binding of MC3 with its antigen, and the inhibition rates were 41.19%, 36.89% and 33.77% respectively. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup and ? type. Conclusion Generation of E. coli HB2151 expressed ScFv of monoclonal antibody MC3 paves the way for further use of the antibody.