data curation; M. a single-chain form of trimeric R1antTNF with a human IgG-Fc domain name. scR1antTNF-Fc experienced properties much like those of R1antTNF, including TNFR1-selective binding avidity, TNFR1 antagonistic activity, and thermal stability, and experienced a significantly extended plasma compared with antibody drugs because it is usually a cytokine-derived structure and therefore its stability and lengthen the dosage interval. Furthermore, the pharmacological effects of this derivative were exhibited experimentally using CIA mice to verify its potential as a therapeutic agent for rheumatoid arthritis. In addition, the effects of scR1antTNF-Fc were compared with that of 40-kDa PEG-scR1antTNF in arthritis mice. Results Fc fusion protein of R1antTNF expressed from mammalian cells We previously reported an designed single-chain structure form of R1antTNF, termed scR1antTNF, which crosslinked three R1antTNF monomers via a GGGSGGG peptide linker (20). The N terminus and C terminus of the R1antTNF monomer are located on the same side as exhibited by crystal structure data (PDB ID 2E7A), and the single-chain structure of R1antTNF OPD2 was very easily generated and properly functioned as a TNFR1 antagonist. In this study, we newly created scR1antTNF-Fc, consisting of scR1antTNF fused to a human IgG1-Fc domain around the C terminus, to extend its and Fig. S1shows an 75-kDa band of the scR1antTNF-Fc monomer. of TNF, scR1antTNF, scR1antTNF-Fc (Vhss), and scR1antTNF-Fc (IL-2ss) were comparable (1.5, 2.6, 4.2, and 4.3 nm, respectively) (Fig. 2of TNF was indicated (4.8 nm), but the of each antagonist was not defined because the binding avidities were too small. These results showed that scR1antTNF-Fcs retained TNFR1-selective binding house after Fc fusion. Open in a separate window Physique 2. binding affinity of scR1antTNF-Fc. receptor-binding ability of human TNF, scR1antTNF, and scR1antTNF-Fcs to human TNFR1 and human TNFR2 was analyzed by SPR. Each sensorgram shows the association (120 s) and dissociation (120 s) repeats at five serial concentrations (1.2, 3.7, 11.1, 33.3, and 100 nm) using single-cycle kinetics. Analytes: TNF, scR1antTNF, scR1antTNF-Fc (Vhss), and scR1antTNF-Fc (IL-2ss). Ligands: Fc chimera proteins of human TNFR1 and human TNFR2. = 1). The avidity of scR1antTNF-Fc (Vhss) and scR1antTNF-Fc (IL-2ss) were analyzed as a bivalent analyte. indicated the dissociation constant, association rate constant, and dissociation rate constant, respectively. The thermal stability of scR1antTNF-Fc was investigated by a thermal shift assay (TSA) using differential scanning fluorometry. TSA can quickly assess the thermal denaturation Chlorpromazine hydrochloride heat (Tm) and Chlorpromazine hydrochloride denaturation process of a protein by increasing fluorescence intensity dependent on structure aggregation. An increase in the peak heat in the TSA indicates an increase in the thermal stability of the protein. A marked peak shift was not observed between scR1antTNF and scR1antTNF-Fcs (Fig. 3values calculated from the result of thermal shift assay using Protein Thermal Shift Software. Bioactivity of scR1antTNF-Fc is usually retained after Fc fusion Bioassays were performed to investigate the effect of scR1antTNF-Fc by Fc fusion. The agonist activity of scR1antTNF-Fc was observed using mouse fibroblast (LM) cells. scR1antTNF originally showed only slight cytotoxicity for LM cells, which preferentially express TNFR1. This indicated that scR1antTNF and scR1antTNF-Fcs function as TNFR1 antagonists. scR1antTNF-Fc (Vhss) and scR1antTNF-Fc (IL-2ss) induced a similar level of cytotoxicity to scR1antTNF (Fig. 4bioactivity of scR1antTNF-Fc via TNFR1 or TNFR2. agonistic bioactivities of scR1antTNF or scR1antTNF-Fcs through TNFR1 for LM cells were measured. Human TNF was used as a control. = 3). The agonistic activity of scR1antTNF-Fc was also assessed by NF-B reporter assay. After the activation of Ramos-Blue Chlorpromazine hydrochloride cells with human TNF, scR1antTNF, or scR1antTNF-Fcs, NF-B-inducible secreted embryonic alkaline phosphatase (SEAP) activity was measured. This assay steps the transmission transduction activity via TNFR1 because Ramos-Blue cells preferentially express TNFR1. TNF increases SEAP activity in a dose-dependent manner, whereas scR1antTNF and scR1antTNF-Fcs induced no activity (Fig. 4stability. Open in a separate window Physique 5. plasma clearance of scR1antTNF-Fc. = 6), etanercept (1250 g/kg) (= 6), and scR1antTNF-Fc (50 g/kg) (= 6) were administered i.p. twice a week from day 22 after immunization. and = 6) in plasma on day 42 was measured by ELISA. Etanercept, 1250 g/kg; scR1antTNF-Fc, 50 g/kg. Data are shown as the mean S.D.; *, 0.05 (one-way ANOVA with Tukey’s multiple comparisons test). T cell populations in the lymph nodes of CIA mice injected with scR1antTNF-Fc scR1antTNF-Fc inhibits TNFR1 signaling selectively. Therefore, TNFR2 signaling is usually transmitted by endogenous TNF. To confirm the advantage of the TNFR1-selective inhibition mechanism, we measured the population of CD4+ or CD8+ T cells in the lymph nodes of saline-, etanercept-, or scR1antTNF-Fc (50 g/kg)Ctreated CIA mice. Lymph node cells were separated to CD8+ T cells and CD4+ T cells, and then CD4+ T cells.