Preparation of a novel EGFR specific immunotoxin and its efficacy of anti-colorectal cancer in vitro and in vivo.

Objectives: Epithelial growth factor receptor (EGFR), as a malignancy marker, is overly expressed in multiple solid tumors including colorectal neoplasms, one of the most prevalent malignancies worldwide. The main objective of this study is to enhance the efficacy of anti-tumor therapy targeting EGFR by constructing a novel EGFR-specific immunotoxin (C-CUS) based on Cetuximab and recombinant Cucurmosin (CUS).

Methods: E. coli BL21 (DE3) PlysS (E. coli) was used to express CUS with a cysteine residue inserting to the C-terminus of Cucurmosin. Then immobilized metal ion affinity chromatography (IMAC) was used to purify CUS. The chemical conjugation method was used for the preparation of C-CUS. Then dialysis and IMAC were used to purify C-CUS. Western blot as well as SDS-PAGE was carried out to characterize the formation of C-CUS. At last the anti-colorectal cancer activity of C-CUS was investigated in vitro and in vivo.

Results: CUS with high purity could be obtained from the prokaryotic system. C-CUS was successfully constructed and highly purified. The cytotoxicity assays in vitro showed a significant proliferation inhibition of C-CUS on EGFR-positive cells for 120 h with IC values less than 0.1 pM. Besides, the anti-tumor efficacy of C-CUS was remarkably more potent than that of Cetuximab, CUS, and C + CUS (P < 0.001). Whereas the cytotoxicity of C-CUS could hardly be detected on EGFR-null cell line. Our results also showed that C-CUS had efficacy of anti-colorectal cancer in mouse xenograft model, indicating the therapeutic potential of C-CUS for the targeted therapy of colorectal neoplasms.

Conclusions: C-CUS exhibits potent and EGFR-specific cytotoxicity. Insertional mutagenesis technique is worthy to be adopted in the preparation of immunotoxin. Immunotoxin can be highly purified through dialysis followed by IMAC.