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

Koki Makabe, Ryutaro Asano, Takahiko Ito, Kouhei Tsumoto, Toshio Kudo, Izumi Kumagai

Research output: Contribution to journalArticle

16 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)98-105
Number of pages8
JournalBiochemical and biophysical research communications
Volume328
Issue number1
DOIs
Publication statusPublished - 2005 Mar 4

Keywords

  • Cancer immunotherapy
  • Fusion protein
  • Fv
  • Interleukin-12
  • Refolding

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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