Tumor-directed lymphocyte-activating cytokines: refolding-based preparation of recombinant human interleukin-12 and an antibody variable domain-fused protein by additive-introduced stepwise dialysis.

Abstract

Integration of lymphocyte-activating cytokines (e.g., interleukin-12: IL-12) to tumor cells offers promise for cancer immunotherapy, but the preparation of such heterodimeric proteins by refolding is difficult because of subunit instability. We achieved the refolding of Escherichia coli-expressed human IL-12 by a stepwise dialysis method, preventing the formation of insoluble aggregates by adding a redox reagent and an aggregation suppressor. We also constructed a tumor-specific IL-12 protein, each subunit of which was fused with one chain of variable domain fragment (Fv) of anticarcinoembryonic antigen (CEA) antibody T84.66 (aCEA-IL12). Fusion of IL-12 with Fv greatly increased the yield of functional heterodimer. Several assays have indicated that the Fv domain and IL-12 domain of the fused protein had cognate biological activities, and it enhanced the cytotoxicity of T-LAK cells for the cancer cell line.

• Authors: Koki Makabe, Ryutaro Asano, Takahiko Ito, Kouhei Tsumoto, Toshio Kudo, Izumi Kumagai
• Journal: Biochemical and biophysical research communications (Biochem Biophys Res Commun) Vol. 328 Issue 1 Pg. 98-105 (Mar 04 2005) ISSN: 0006-291X [Print] United States
• PMID: 15670756 (Publication Type: Journal Article)

Chemical References

• Antibodies
• Carcinoembryonic Antigen
• Cytokines
• Recombinant Fusion Proteins
• Interleukin-12

Topics

• Animals
• Antibodies (administration & dosage, genetics, immunology)
• Bile Duct Neoplasms (drug therapy, immunology)
• Carcinoembryonic Antigen (administration & dosage, genetics, immunology)
• Cell Line, Tumor
• Cell Proliferation (drug effects)
• Cytokines (administration & dosage, chemistry, genetics, immunology)
• Dose-Response Relationship, Drug
• Humans
• Interleukin-12 (administration & dosage, chemistry, genetics, immunology)
• Lymphocyte Activation (drug effects)
• Microdialysis (methods)
• Protein Engineering (methods)
• Protein Folding
• Recombinant Fusion Proteins (administration & dosage, chemistry)