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.
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.
Makabe
Koki
K
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Aoba-ku, Sendai 980-8579, Japan.
Asano
Ryutaro
R
Ito
Takahiko
T
Tsumoto
Kouhei
K
Kudo
Toshio
T
Kumagai
Izumi
I
D000818:Animals; D000906:Antibodies / Q000008:administration & dosage* / Q000235:genetics / Q000276:immunology; D001650:Bile Duct Neoplasms / Q000188:drug therapy* / Q000276:immunology; D002272:Carcinoembryonic Antigen / Q000008:administration & dosage* / Q000235:genetics / Q000276:immunology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D016207:Cytokines / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D018664:Interleukin-12 / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008213:Lymphocyte Activation / Q000187:drug effects*; D017551:Microdialysis / Q000379:methods; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000737:chemistry
D000906:Antibodies; D002272:Carcinoembryonic Antigen; D016207:Cytokines; D011993:Recombinant Fusion Proteins; D018664:Interleukin-12