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
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
UR - http://www.scopus.com/inward/record.url?scp=12844258956&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=12844258956&partnerID=8YFLogxK
U2 - 10.1016/j.bbrc.2004.12.141
DO - 10.1016/j.bbrc.2004.12.141
M3 - Article
C2 - 15670756
AN - SCOPUS:12844258956
VL - 328
SP - 98
EP - 105
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 1
ER -