The Construction Of Engineered And Energized Fusion Proteins Consisting Of Lidamycin And VH/VL Single Domain Of Anti-Type Ⅳ Collagenase Antibody And Their Antitumor Activity

Successful development of tumor-targeted therapeutic agents is dependent, in part, on the site-specific delivery of therapeutic agents and on the biological activity of the delivered agent. MAbs have been used to impart selectivity to otherwise indiscriminately cytotoxic agents such as toxins, radionuclides. However, clinical use of mAb-based agents has met with limited success due to the huge size of intact mAbs. Murine mAb 3G11 can specifically bind to type IV collagenase, and some targeted drugs based on 3G11 have led to some striking antitumor effects. To reduce the molecular weight of immunoconjugate and obtain smaller agents for targeted immunotherapy of cancer, in this study, we constructed both single-chain Fv fragment of mAb 3G11 directed against Type IV collagenase and energized fusion proteins consisting of LDM and VH/VL single domain antibodies derived from 3G11, and further studied their biological activity.1. Expression, purification, refolding and characterization of single-chain Fv fragment directed against type IV collagenase produced in Escherichia coliType IV collagenase is a critical proteolytic enzyme involving in tumor invasion and metastasis. Inhibition of type IV collagenase activity results in anti-metastatic activity. In the experiment, we amplified the scFv gene by PCR introducing restriction sites and cloned it into the vector pET-30a(+) under the control of T7lac promotor. The resulting recombinant plasmid named pET-scFv was transformed into E. coli BL21star (DE3) and expression of the recombinant scFv gene was induced by addition of 0.2 mM IPTG. The yield of expressed proteins mainly as insoluble inclusion bodies was about 30% of total cellular proteins. After purification by IMAC under denatured conditions, the scFv fragments were refolded in a way of step-wise dialysis. Approximately 30 mg soluble functional scFv could be harvested from one liter fermentation broth, and the purity of the scFv exceeded 95%. The purified and refolded 3G11-scFv exhibited significant hapten binding activity by ELISA, the association constant with type IV collagenase is 6×10⁷M^-1. The 3G11-scFv can specifically bind to different kinds of tumor cells such as KB,...