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

Abstract

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-CUS245C) based on Cetuximab and recombinant Cucurmosin (CUS245C).

METHODS:

E. coli BL21 (DE3) PlysS (E. coli) was used to express CUS245C with a cysteine residue inserting to the C-terminus of Cucurmosin. Then immobilized metal ion affinity chromatography (IMAC) was used to purify CUS245C. The chemical conjugation method was used for the preparation of C-CUS245C. Then dialysis and IMAC were used to purify C-CUS245C. Western blot as well as SDS-PAGE was carried out to characterize the formation of C-CUS245C. At last the anti-colorectal cancer activity of C-CUS245C was investigated in vitro and in vivo.

RESULTS:

CUS245C with high purity could be obtained from the prokaryotic system. C-CUS245C was successfully constructed and highly purified. The cytotoxicity assays in vitro showed a significant proliferation inhibition of C-CUS245C on EGFR-positive cells for 120 h with IC50 values less than 0.1 pM. Besides, the anti-tumor efficacy of C-CUS245C was remarkably more potent than that of Cetuximab, CUS245C, and C + CUS245C (P < 0.001). Whereas the cytotoxicity of C-CUS245C could hardly be detected on EGFR-null cell line. Our results also showed that C-CUS245C had efficacy of anti-colorectal cancer in mouse xenograft model, indicating the therapeutic potential of C-CUS245C for the targeted therapy of colorectal neoplasms.

CONCLUSIONS:

C-CUS245C 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.

• Authors: Shuifa Wu, Cuimin Deng, Caiyun Zhang, Jiani Xiong, Xiaofan Gu, Ze Wang, Jingjing Tu, Jieming Xie
• Journal: Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico (Clin Transl Oncol) Vol. 23 Issue 8 Pg. 1549-1560 (Aug 2021) ISSN: 1699-3055 [Electronic] Italy
• PMID: 33474678 (Publication Type: Journal Article)

Chemical References

• Antineoplastic Agents, Immunological
• Immunoconjugates
• Immunotoxins
• Plant Proteins
• cucurmosin protein, Cucurbita moschata
• EGFR protein, human
• ErbB Receptors
• Cetuximab

Topics

• Animals
• Antineoplastic Agents, Immunological (pharmacology)
• Cell Line, Tumor
• Cell Proliferation (drug effects)
• Cetuximab (pharmacology, therapeutic use)
• Chromatography, Affinity (methods)
• Colorectal Neoplasms (metabolism, pathology, therapy)
• ErbB Receptors (antagonists & inhibitors, metabolism)
• Escherichia coli (metabolism)
• Humans
• Immunoconjugates (chemistry, therapeutic use)
• Immunotoxins (chemistry, isolation & purification, metabolism, therapeutic use)
• Male
• Mice
• Mice, Inbred BALB C
• Mice, Nude
• Molecular Targeted Therapy (methods)
• Mutagenesis, Insertional (methods)
• Plant Proteins (chemistry, metabolism, therapeutic use)
• Time Factors
• Xenograft Model Antitumor Assays