RRC ID 55910
著者 Osaki T, Nakanishi T, Aoki M, Omizu T, Nishiura D, Kitamura M.
タイトル Soluble Expression in Escherichia coli of a Single-Domain Antibody-Tumor Necrosis Factor α Fusion Protein Specific for Epidermal Growth Factor Receptor.
ジャーナル Monoclon Antib Immunodiagn Immunother
Abstract Tumor-targeted antibody-cytokine fusion proteins, called immunocytokines, are expected to be a useful platform for the development of effective antitumor therapeutic agents; however, their design and cost-efficient production remain as challenges. In this study, we constructed an antibody-cytokine fusion protein (Ia1-TNFα) comprising the single-domain antibody Ia1, which targets epidermal growth factor receptor (EGFR) overexpressed in epithelial tumors and a tumor necrosis factor α (TNFα) domain, which has antitumor activity. Ia1-TNFα was produced in a soluble form by using an Escherichia coli expression system, and after affinity purification of the culture supernatant, an yield of ∼2 mg/L of cell culture was obtained. Gel filtration analysis showed that Ia1-TNFα existed predominantly as a trimer, which is consistent with the multimerization state of TNFα. Ia1-TNFα exhibited approximately 7-fold lower TNFα biological activity than that of TNFα itself. Flow cytometric analysis revealed that Ia1-TNFα specifically bound to EGFR-expressing tumor cells and that its binding activity was higher than that of monovalent Ia1, suggesting that the fusion protein bound to the tumor cells multivalently. Altogether, these results show that fusion of TNFα with a single-domain antibody could be a cost-efficient means of producing antitumor therapeutic agents.
巻・号 37(1)
ページ 20-25
公開日 2018-2-1
DOI 10.1089/mab.2017.0051
PMID 29474158
MeSH Antineoplastic Agents / chemistry Antineoplastic Agents / pharmacology* Carcinoma, Squamous Cell / drug therapy* Carcinoma, Squamous Cell / metabolism Carcinoma, Squamous Cell / pathology Cell Proliferation / drug effects ErbB Receptors / antagonists & inhibitors* Escherichia coli / genetics Escherichia coli / metabolism* Humans Recombinant Fusion Proteins / chemistry Recombinant Fusion Proteins / pharmacology* Single-Chain Antibodies / chemistry* Tumor Cells, Cultured Tumor Necrosis Factor-alpha / chemistry*
リソース情報
ヒト・動物細胞 A431(RCB0202)