TY - JOUR
T1 - Tumor-directed lymphocyte-activating cytokines
T2 - Refolding-based preparation of recombinant human interleukin-12 and an antibody variable domain-fused protein by additive-introduced stepwise dialysis
AU - Makabe, Koki
AU - Asano, Ryutaro
AU - Ito, Takahiko
AU - Tsumoto, Kouhei
AU - Kudo, Toshio
AU - Kumagai, Izumi
N1 - Funding Information:
This work was supported in part by Grants-in-Aid (R.A., K.T., and I.K.) from the Japan Society for the Promotion of Science and the Ministry of Education, Science, Sports and Culture of Japan. Support was also provided through the Proposal-Based Research and Development Promotion Program and the Industrial Technology Research Grant Program of the New Energy and Industrial Technology Development Organization (NEDO) of Japan.
PY - 2005/3/4
Y1 - 2005/3/4
N2 - 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.
AB - 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.
KW - Cancer immunotherapy
KW - Fusion protein
KW - Fv
KW - Interleukin-12
KW - Refolding
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U2 - 10.1016/j.bbrc.2004.12.141
DO - 10.1016/j.bbrc.2004.12.141
M3 - Article
C2 - 15670756
AN - SCOPUS:12844258956
SN - 0006-291X
VL - 328
SP - 98
EP - 105
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 1
ER -
