Abstract: AIM　To develop a novel, highly potent antitumor immunoconjugate of smaller molecular size by linking lidamycin (also called C1027), an antibiotic with extremely potent cytotoxicity to cancer cells, to an scFv antibody directed against type IV collagenase. METHODS　Lidamycin (LDM) molecule consists of an enediyne chromophore (LDC) and a protein (LDP) of 10.5 ku. The two moieties of the molecule can be separated and an active molecule can be reconstituted. The novel assembled fusion protein, an antitumor immunoconjugate, was manufactured through genetic engineering and assembled molecule reconstitution. Firstly, a recombinant fusion protein LDP-Fv was obtained from genetically engineered E.coli JM109-pKFL. Secondly, the assembled fusion protein LDM-Fv was prepared by adding LDC to LDP-Fv. RESULTS　By 12% SDS-PAGE the E.coli-expressed fusion protein LDP-Fv showed a distinct band of approximately 37 ku accounting for about 8% of the total protein. LDP-Fv, which was non-cytotoxic, retained the immunoreactivity of the original monoclonal antibody directed against type IV collagenase. LDV-Fv significantly reduced the activity of type IV collagenase in highly metastatic human lung carcinoma PG cells and inhibited cell invasion in dose-dependent manner. After molecule reconstitution, the assembled fusion protein LDM-Fv showed extremely potent cytotoxicity to PG cells with an IC₅₀ value of 9.5×10^-15 mol.L^-1 by clonogenic assay. CONCLUSION The engineered and assembled fusion protein LDM-Fv is a highly effective antitumor immunoconjugate with much smaller molecular size than those ever reported.