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The Journal of Biological Chemistry logoLink to The Journal of Biological Chemistry
. 2020 May 12;295(28):9379–9391. doi: 10.1074/jbc.RA120.012723

Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics

Masaki Inoue 1,2,3, Yuta Tsuji 1, Chinatsu Yoshimine 1, Shota Enomoto 1, Yuki Morita 1, Natsuki Osaki 1, Masahiro Kunishige 1, Midori Miki 1, Shota Amano 1, Kanako Yamashita 1, Haruhiko Kamada 2,3,4, Yasuo Tsutsumi 4,5, Shin-ichi Tsunoda 1,2,3,4,*
PMCID: PMC7363114  PMID: 32398258

Abstract

Excessive activation of the proinflammatory cytokine tumor necrosis factor-α (TNFα) is a major cause of autoimmune diseases, including rheumatoid arthritis. TNFα induces immune responses via TNF receptor 1 (TNFR1) and TNFR2. Signaling via TNFR1 induces proinflammatory responses, whereas TNFR2 signaling is suggested to suppress the pathophysiology of inflammatory diseases. Therefore, selective inhibition of TNFR1 signaling and preservation of TNFR2 signaling activities may be beneficial for managing autoimmune diseases. To this end, we developed a TNFR1-selective, antagonistic TNFα mutant (R1antTNF). Here, we developed an R1antTNF derivative, scR1antTNF-Fc, which represents a single-chain form of trimeric R1antTNF with a human IgG-Fc domain. scR1antTNF-Fc had properties similar to those of R1antTNF, including TNFR1-selective binding avidity, TNFR1 antagonistic activity, and thermal stability, and had a significantly extended plasma t1/2 in vivo. In a murine rheumatoid arthritis model, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF (a previously reported PEGylated form) delayed the onset of collagen-induced arthritis, suppressed arthritis progression in mice, and required a reduced frequency of administration. Interestingly, with these biologic treatments, we observed an increased ratio of regulatory T cells to conventional T cells in lymph nodes compared with etanercept, a commonly used TNF inhibitor. Therefore, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF indirectly induced immunosuppression. These results suggest that selective TNFR1 inhibition benefits the management of autoimmune diseases and that R1antTNF derivatives hold promise as new-modality TNF-regulating biologics.

Keywords: tumor necrosis factor (TNF), cytokine, autoimmune disease, arthritis, inflammation, inhibition mechanism, protein engineering, drug design, drug delivery, forkhead box P3 (FOXP3), antagonist, single-chain

Biologics represented by antibody drugs have dramatically changed the way of treating autoimmune diseases, including rheumatoid arthritis, psoriasis, and ulcerative colitis. For example, in rheumatoid arthritis, treatment with disease-modifying anti-rheumatic drugs have shifted to treatment with antibody drugs that target TNFα or IL-6 receptor (IL-6R) with superior efficacy, which has led to an improved quality of life for patients (1, 2). However, some patients with rheumatoid arthritis are intolerant of these antibody drugs or may develop inadequate responses or loss of response over time (3). Furthermore, clinical reports showed that switching to different anti-TNF drugs is effective when the first drug has failed or resulted in intolerance (4, 5). Therefore, biologics with new efficacy or mechanisms are being developed and their presence in clinical practice will increase in the future.

TNFα is a cytokine involved in the onset and deterioration of autoimmune diseases. It mediates different physiological functions through two receptor subtypes, TNFR1 and TNFR2. TNFR1 signaling is involved in inflammatory responses and affects the pathology of immune disorders. However, TNFR2 signaling is considered to have a protective role, including neuroprotection (6) and defense against viral infection (7, 8). TNFR2 signaling was recently suggested to induce the proliferation and activation of regulatory T cells (Tregs) responsible for immunosuppression (9–11). At present, antibody drugs that neutralize TNFα have shown excellent therapeutic effects in the treatment of immunogenic disorders. However, because they inhibit intracellular signaling through TNFR1 and TNFR2, the use of TNFR1-selective inhibition strategies might be more effective (12, 13). Indeed, therapeutics and biologics focused on the function of specific TNF receptor signaling pathways are expected for multiple disorders (14–16).

Therefore, to develop anti-TNF drugs with new modalities, we created a TNFα variant protein R1antTNF that functions as a TNFR1 antagonist and that selectively inhibits TNFR1 signaling involved in the pathogenesis of immunogenic disorders (17). R1antTNF specifically binds to TNFR1, but not TNFR2, and does not transmit signals via TNFR1. Thus, R1antTNF competes with TNF at TNFR1. We reported the pharmacological effects of R1antTNF in a mouse model of rheumatoid arthritis (18) and multiple sclerosis (19). R1antTNF modified with 5-kDa PEG (5-kDa PEG-R1antTNF) suppressed arthritis in collagen-induced arthritis (CIA) mice (18). In addition, 5-kDa PEG-R1antTNF decreased the paralysis of limbs and tails in experimental autoimmune encephalomyelitis mice (19). However, R1antTNF has poor stability in vivo compared with antibody drugs because it is a cytokine-derived structure and therefore its t1/2 in the blood is short even with 5-kDa PEGylation (18). Therefore, we generated a single-chain R1antTNF (scR1antTNF), where the trimeric structure of R1antTNF is fused with a peptide linker as a single-chain structure to improve its molecular stability and molecular-modification efficiency (20). As the result, mono-PEGylated scR1antTNF with 40-kDa PEG (40-kDa PEG-scR1antTNF) was easily generated and its t1/2 in blood was significantly extended.

In this study, we investigated the further structural optimization of a TNFR1-selective antagonist protein. The R1antTNF derivative with a human IgG-Fc domain, termed scR1antTNF-Fc, was created to enhance its in vivo stability and extend the dosage interval. Furthermore, the pharmacological effects of this derivative were demonstrated 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 engineered 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 demonstrated by crystal structure data (PDB ID 2E7A), and the single-chain structure of R1antTNF was 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 on the C terminus, to extend its t1/2 in vivo. The model structure of scR1antTNF-Fc demonstrated that TNFR1 antagonist proteins become bivalent by fusion with the human IgG1-Fc domain via a hinge region (Fig. 1A and Fig. S1A). The Fc domain did not affect the binding ability of the scR1antTNF domain to TNFR1, because the Fc proteins were located on the top of scR1antTNF, which is different from the receptor interaction region (Fig. 1B). pCAG-based expression vectors for scR1antTNF-Fc (Fig. 1C) were engineered and transfected into Expi293F cells. Two types of secretion sequences derived from human IgG heavy chain (Vhss) or human IL-2 (IL-2ss) were used to confirm superior protein secretion and expression (21). After cell cultivation for 7 days, scR1antTNF-Fc expression was confirmed by SDS-PAGE (Fig. 1D, left). The band of each protein expressed with Vhss or IL-2ss was detected at 75 kDa as a monomer of similar density. The recombinant proteins were then purified using immobilized metal ion affinity chromatography and size-exclusion chromatography. scR1antTNF-Fc fused to Vhss and IL-2ss were detected as a single peak with a similar retention time and good purity (Fig. 1E). Purification was also confirmed by a single band at the expected molecular weight by SDS-PAGE (Fig. 1D, right). Moreover, Western blotting showed that both scR1antTNF-Fcs co-migrated as a single band with an apparent molecular mass of about 75 kDa (Fig. 1F). Thus, it was confirmed that scR1antTNF-Fc forms an Fc fusion protein with scR1antTNF consisting of two 75-kDa monomers similar to IgG.

Figure 1.

Figure 1.

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Generation and characterization of scR1antTNF-Fc protein. A, schematic structure modeling of scR1antTNF-Fc protein. Two TNFR1 antagonistic proteins were fused with human IgG-Fc. N, N-terminal. Amino acid sequences and domain information of scR1antTNF-Fc are described in Fig. S1A. B, X-ray structure modeling of the scR1antTNF-TNFR1 complex. scR1antTNF bound to the homotrimer of TNFR1. Red, TNFR1; green, TNFR1 interaction domain of scR1antTNF; orange, peptide linker for forming the single-chain structure. C, schematic pCAG-based mammalian expression vector for scR1antTNF-Fc protein. The cDNA was composed to express scR1antTNF-Fc whereby triple R1antTNF domains fused by peptide linkers (GGGSGGG) were further fused to a human–IgG Fc domain (Ch2 and Ch3). Signal sequence peptide genes derived from a mouse IgG Vh (Vhss) or human IL-2 (IL-2ss) were linked at the 5′-terminal of scR1antTNF-Fc cDNA. D, supernatants of cultured medium (left side) and purified proteins (right side) 7 days after transfection were assessed by SDS-PAGE following Coomassie Brilliant Blue staining. Arrowhead shows an ∼75-kDa band of the scR1antTNF-Fc monomer. E, each recombinant protein expressed with Vhss and IL-2ss was purified by size-exclusion chromatography. F, the molecular weight of monomeric scR1antTNF-Fc protein was confirmed by Western blotting with an anti-human IgG-Fc antibody.

In vitro receptor selectivity and thermal stability of scR1antTNF-Fc are retained when an Fc domain is fused

TNFα binds to TNFR1 and TNFR2, but scR1antTNF binds only to TNFR1 (20). The binding avidities of scR1antTNF-Fc (Vhss) and scR1antTNF-Fc (IL-2ss) against TNF receptors were assessed using surface plasmon resonance (SPR) to confirm its binding specificity for TNFR1 (Fig. 2A). The affinities of TNFα and scR1antTNF and the avidities of scR1antTNF-Fcs were evaluated at concentrations of 6.25, 12.5, 25.0, 50.0, and 100 nm by single-cycle kinetics. scR1antTNF-Fc (Vhss) and scR1antTNF-Fc (IL-2ss) showed dose-dependent responses for TNFR1 as well as scR1antTNF. At a maximum concentration of each protein, maximum binding response (Rmax) values were obtained from the sensorgram for TNFR1 (TNFα, scR1antTNF, scR1antTNF-Fc (Vhss), scR1antTNF-Fc (IL-2ss); 135, 32.3, 78.8, 82.2 RU, respectively). TNFα bound to TNFR2 (Rmax: 27.6 RU). However, scR1antTNF-Fc (Vhss) and scR1antTNF-Fc (IL-2ss) had poorer binding ability to TNFR2 as well as scR1antTNF (Fig. 2A). Thus, these scR1antTNF-Fcs appeared to selectively interact with TNFR1 in preference to TNFR2. From these sensorgrams, the dissociation constants (Kd) were calculated. The TNFR1 Kd of TNFα, scR1antTNF, scR1antTNF-Fc (Vhss), and scR1antTNF-Fc (IL-2ss) were similar (1.5, 2.6, 4.2, and 4.3 nm, respectively) (Fig. 2B). The TNFR2 Kd of TNFα was indicated (4.8 nm), but the Kd of each antagonist was not defined because the binding avidities were too small. These results showed that scR1antTNF-Fcs retained TNFR1-selective binding property after Fc fusion.

Figure 2.

Figure 2.

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In vitro binding affinity of scR1antTNF-Fc. A, in vitro receptor-binding ability of human TNFα, 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: TNFα, scR1antTNF, scR1antTNF-Fc (Vhss), and scR1antTNF-Fc (IL-2ss). Ligands: Fc chimera proteins of human TNFR1 and human TNFR2. B, kinetic parameters of each protein to human TNFR1/TNFR2 were analyzed with a 1:1 binding model using BIAcore × 100 evaluation software (n = 1). The avidity of scR1antTNF-Fc (Vhss) and scR1antTNF-Fc (IL-2ss) were analyzed as a bivalent analyte. Kd, ka, and kd indicated the dissociation constant, association rate constant, and dissociation rate constant, respectively.

The thermal stability of scR1antTNF-Fc was investigated in vitro by a thermal shift assay (TSA) using differential scanning fluorometry. TSA can quickly assess the thermal denaturation temperature (Tm) and denaturation process of a protein by increasing fluorescence intensity dependent on structure aggregation. An increase in the peak temperature 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. 3A). From these results, the Tm value of scR1antTNF was determined to be 75.7 ± 0.2°C (Fig. 3B). By contrast, scR1antTNF-Fc (Vhss) and scR1antTNF-Fc (IL-2ss) showed biphasic peaks (Fig. 3A), and tandem Tm values were 69.4 ± 0.1°C/75.8 ± 0.1°C and 70.1 ± 0.1°C/75.6 ± 0.2°C, respectively. Because the Tm value of etanercept was 69.1 ± 0.1°C, the smaller biphasic peaks of scR1antTNF-Fc might indicate that denaturing begins in the Fc region. Therefore, no significant difference in the thermal stability of the TNFR1 antagonist domain was found between scR1antTNF and scR1antTNF-Fc, even after Fc fusion. These results indicate that scR1antTNF-Fc has high thermal stability as well as anti-TNF properties.

Figure 3.

Figure 3.

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Thermal stability of scR1antTNF-Fc. A, thermal stabilities of scR1antTNF, scR1antTNF-Fc (Vhss), scR1antTNF-Fc (IL-2ss) and etanercept were measured by thermal shift assay using differential scanning fluorometry. Proteins serially diluted from 250 μg/ml by 2-fold dilution are indicated. B, temperature of the peak apex of five concentrations show the denaturation temperature (Tm). Tm values calculated from the result of thermal shift assay using Protein Thermal Shift Software.

Bioactivity of scR1antTNF-Fc is 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. 4A). Furthermore, antagonistic activity was observed. scR1antTNF suppressed TNFα-induced LM cell death. scR1antTNF-Fcs also induced TNFα inhibitory activity in a dose-dependent manner, and the activity tended to be higher than that of scR1antTNF (Fig. 4B). No difference in cytotoxic activity between IgG-Vhss and IL-2ss was observed. Therefore, these results indicated that the antagonistic activities of scR1antTNF-Fc were retained by the Fc fusion.

Figure 4.

Figure 4.

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In vitro bioactivity of scR1antTNF-Fc via TNFR1 or TNFR2. A, in vitro agonistic bioactivities of scR1antTNF or scR1antTNF-Fcs through TNFR1 for LM cells were measured. Human TNFα was used as a control. B, antagonistic activities of scR1antTNF, scR1antTNF-Fc (Vhss), and scR1antTNF-Fc (IL-2ss) via TNFR1 were confirmed by LM cell assay. LM cells were treated with each protein in the presence of mouse TNFα (5 ng/ml). The TNF inhibition rate was determined from the LM cell viability. C, NF-κB induction was evaluated by reporter assay using Ramos-Blue cells. Ligand-dependent SEAP activities were detected. D, agonistic activity via TNFR2 was evaluated by the cell death of huTNFR2/mFas preadipocytes. Data are shown as the mean ± S.D. (n = 3).

The agonistic activity of scR1antTNF-Fc was also assessed by NF-κB reporter assay. After the stimulation of Ramos-Blue cells with human TNFα, scR1antTNF, or scR1antTNF-Fcs, NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) activity was measured. This assay measures the signal transduction activity via TNFR1 because Ramos-Blue cells preferentially express TNFR1. TNFα increases SEAP activity in a dose-dependent manner, whereas scR1antTNF and scR1antTNF-Fcs induced no activity (Fig. 4C). This confirmed that the TNFR1 antagonist Fc fusion protein did not have agonistic activity by activating NF-κB signaling through TNFR1. In addition, a cytotoxic assay using huTNFR2/mFas-preadipocytes was used to confirm these bioactivities via TNFR2 (Fig. 4D). We found that cells overexpressing a fusion chimera receptor of human TNFR2 and mouse Fas induced cell death by TNFR2 stimulation (22). As a result, TNFα displayed concentration-dependent cytotoxicity, but scR1antTNF induced slight cell death as reported previously (20). Interestingly, scR1antTNF-Fcs induced concentration-dependent cytotoxicity with a lower activity than TNFα. Therefore, scR1antTNF-Fcs seem to exert agonistic activity via TNFR2 because they induce TNFR2 signal transduction similar to membrane-bound TNF.

Plasma clearance of scR1antTNF-Fc is extended by Fc fusion

To evaluate the stability of scR1antTNF-Fc in vivo, plasma clearance was measured after a single intraperitoneal administration to mice (Fig. 5A). scR1antTNF-Fc had the same amino acid sequence regardless of which expression vector (Vhss or IL-2ss) was used, because the signal sequence was cleaved. Indeed, both scR1antTNF-Fcs had similar kinetics and bioactivities. Therefore, we used scR1antTNF-Fc (Vhss) to assess its pharmacokinetics. The t1/2 of scR1antTNF was 4.1 h, and it disappeared from the blood within 24 h after administration. By contrast, plasma concentrations of scR1antTNF-Fc peaked 90 min after i.p. injection and were maintained for a week. The concentration of scR1antTNF-Fc was higher than that of etanercept. The t1/2 of scR1antTNF-Fc was 193.4 h, which was extended compared with etanercept (131.3 h) (Fig. 5B). The area under the curve (AUC) of scR1antTNF was more than twice that of etanercept. These results indicated that the Fc fusion protein of scR1antTNF-Fc dramatically increased its in vivo stability.

Figure 5.

Figure 5.

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In vivo plasma clearance of scR1antTNF-Fc. A, plasma clearances of scR1antTNF, scR1antTNF-Fc, and etanercept were confirmed after i.p. injection. Etanercept was used as a positive control. Plasma concentrations of these proteins were measured by ELISA for human TNF or human IgG-Fc. Data are shown as the mean ± S.D. of five mice per group. B, half-lives and AUCs were calculated from time-concentration curves by moment analysis.

scR1antTNF-Fc treatment inhibits the induction of CIA

To evaluate the effects of scR1antTNF-Fc treatment on the development of arthritis, we investigated the severity of CIA mice treated with saline, etanercept (1250 μg/kg), and scR1antTNF-Fc (50 μg/kg) twice a week for 3 weeks (Fig. 6A). CIA is a chronic autoimmune model of human rheumatoid arthritis that is widely used for evaluating potential therapeutic agents. Etanercept was used as a positive control. Scoring the degree of arthritis demonstrated a significant decrease in arthritis in scR1antTNF-Fc (50 μg/kg)–treated mice compared with saline-treated mice (Fig. 6B). The arthritis suppressive effect of scR1antTNF-Fc was similar to that of etanercept. Weight decrease was not observed in scR1antTNF-Fc (50 μg/kg)–, saline-, or etanercept-treated mice (Fig. 6C). The incidence of disease in scR1antTNF-Fc (50 μg/kg)– and etanercept-treated mice was consistently lower than that of saline-treated mice (Fig. 6D). At 42 days after immunization, swelling of hind limbs in saline-treated mice was severe compared with scR1antTNF-Fc (50 μg/kg)– or etanercept-treated mice (Fig. 6E). These results indicate that scR1antTNF-Fc (50 μg/kg) treatment effectively suppressed arthritis in CIA mice.

Figure 6.

Figure 6.

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scR1antTNF-Fc treatment suppresses inflammation in arthritis mice. A, DBA/1 mice were immunized by the subcutaneous injection of bovine type II collagen with CFA. Saline (n = 6), etanercept (1250 μg/kg) (n = 6), and scR1antTNF-Fc (50 μg/kg) (n = 6) were administered i.p. twice a week from day 22 after immunization. B and C, sum of arthritis scores of four paws (B) and body weight (C) were measured for 3 weeks. D, arthritis incidence was calculated from the number of mice with swollen limbs. E, joint swelling in a representative individual from each group at day 42 is shown. Data are shown as the mean ± S.E.; *p < 0.05 (one-way ANOVA with Tukey's multiple comparisons test).

Histopathologic features of joints and inflammatory cytokine levels in the blood of CIA mice treated with scR1antTNF-Fc

H&E staining to investigate the histopathologic features of swollen joints in saline-treated mice indicated that joint cavities were filled with inflammatory cells such as mononuclear cells and neutrophils and a substantial thickening of the synovium, which has also been observed in human rheumatoid arthritis patients (Fig. 7A). In contrast, these pathological features were suppressed in etanercept (1250 μg/kg)- or scR1antTNF-Fc (50 μg/kg)–treated mice at 42 days after immunization. The results of grading right front or right hind paws also indicated that the arthritis states (cell infiltration, synovitis, destruction of cartilage, the juxta-articular bone involvement) were suppressed in etanercept (1250 μg/kg)- or scR1antTNF-Fc (50 μg/kg)–treated mice (Fig. 7B). Tartrate-resistant acid phosphatase (TRAP) staining data in limb joint tissues showed TRAP-positive cells were frequently detected in saline-treated mice at day 42 (Fig. 7C). Osteoclasts express high amounts of TRAP. TRAP-positive cells in rheumatoid synovium may induce the destruction of articular cartilage. However, scR1antTNF-Fc (50 μg/kg) and etanercept (1250 μg/kg) suppressed the number of TRAP-positive cells in the joints of limbs. To examine whether scR1antTNF-Fc treatment affected inflammatory cytokines in vivo, we confirmed IL-1β levels in plasma recovered from the orbital sinus of anesthetized mice at 42 days after immunization. Increased IL-1β levels in saline-treated mice were suppressed in scR1antTNF-Fc (50 μg/kg)– and etanercept-treated mice (Fig. 7D).

Figure 7.

Figure 7.

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Effects of scR1antTNF-Fc treatment on joint pathology and blood cytokine levels. A, after treatment of CIA mice with saline, etanercept (1250 μg/kg), or scR1antTNF-Fc (50 μg/kg) for 3 weeks (on day 42), histological sections of the ankle joint from a hind limb were prepared and stained with H&E. B, histopathologic features such as cell infiltration, synovitis, destruction of cartilage, and the juxta-articular bone involvement on day 42 were scored. C, TRAP-positive cells were stained using serial sections to detect osteoclasts. TRAP-positive cells with ≥1 nucleus were counted as indicated by an arrow. D, mouse IL-1β (n = 6) in plasma on day 42 was measured by ELISA. Etanercept, 1250 μg/kg; scR1antTNF-Fc, 50 μg/kg. Data are shown as the mean ± S.D.; *, p < 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 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 μg/kg)–treated CIA mice. Lymph node cells were separated to CD8+ T cells and CD4+ T cells, and then CD4+ T cells were further fractionated to CD4+ forkhead box P3 (Foxp3)+ Treg cells and CD4+ Foxp3− conventional T cells (Tconvs) (Fig. 8A). Statistical analyses indicated no significant differences in the percentages of CD4+ or CD8+ T cells among the three groups (Fig. 8B). However, numbers of CD4+ Foxp3+ Tregs in scR1antTNF-Fc–treated mice were significantly higher than in saline- or etanercept-treated mice (Fig. 8C). Furthermore, there were fewer CD4+ Foxp3− Tconvs in scR1antTNF-Fc–treated mice. Therefore, the ratio of Tregs to Tconvs was significantly increased in scR1antTNF-Fc–treated mice. The balance between CD4+ Foxp3+ Tregs and CD4+ Foxp3− Tconvs was biased to the Tregs side by the administration of scR1antTNF-Fc. It was reported that TNFR2 was preferentially expressed in Tregs. These results showed that TNFR1-selective inhibition might increase Treg induction by retaining TNFR2 signaling.

Figure 8.

Figure 8.

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Effect of scR1antTNF-Fc administration on T cell subpopulations. At day 42, lymph nodes were isolated from CIA mice administered i.p. with saline (n = 6), etanercept (1250 μg/kg) (n = 6), or scR1antTNF-Fc (50 μg/kg) (n = 6) for 3 weeks. After single cells were prepared, the expressions of lymphocyte markers were analyzed by flow cytometry. A, representative flow cytometry data of each administration group are shown. T cells were separated by CD8, CD4, and Foxp3 expression levels. B, the percentage of CD4+ T cells and CD8+ T cells in lymph node cells was analyzed. C, the percentage of CD4+ Foxp3+ Tregs and CD4+ Foxp3− Tconvs in CD4+ T cells was measured. The ratio of Tregs/Tconvs was calculated from the results of individual mice. Data are shown as the mean ± S.D. of eight mice per group; *, p < 0.05, **, p < 0.01 (one-way ANOVA with Tukey's multiple comparisons test).

PEGylated scR1antTNF also suppresses arthritis in CIA mice

We reported 40-kDa PEG-scR1antTNF in our previous study (20). This molecule consists of scR1antTNF linked with a 40-kDa branched PEG chain at the N-terminal (Fig. 9A and Fig. S1B). 40-kDa PEG-scR1antTNF exerted antagonistic activity for TNFR1 similar to scR1antTNF. However, the molecular weight of 40-kDa PEG-scR1antTNF (∼100 kDa) was lower than that of scR1antTNF-Fc (∼150 kDa). Therefore, there may be some differences in terms of distribution or excretion between scR1antTNF-Fc and 40-kDa PEG-scR1antTNF. We investigated the suppressive effects of arthritis with scR1antTNF-Fc and 40-kDa PEG-scR1antTNF in CIA mice. Saline, etanercept (1250 μg/kg), scR1antTNF-Fc (10 μg/kg), and 40-kDa PEG-scR1antTNF (10 μg/kg) were administrated to CIA mice twice a week from day 22 after immunization. The dose of scR1antTNF-Fc was one fifth of the amount in the above section. A significant decrease of arthritis score was observed in scR1antTNF-Fc (10 μg/kg)– and 40-kDa PEG-scR1antTNF (10 μg/kg)–treated mice as well as etanercept (1250 μg/kg)–treated mice (Fig. 9B). However, no significant weight loss was observed in these groups (Fig. 9C). At day 35 after immunization, swelling of front limbs was not observed in scR1antTNF-Fc–, 40-kDa PEG-scR1antTNF–, and etanercept-treated mice, but it was severe in saline-treated mice (Fig. 9D). Furthermore, the TNFR1-selective inhibition effects of 40-kDa PEG-scR1antTNF treatment were confirmed. There were no differences in the populations of CD4+ or CD8+ T cells in lymph nodes from CIA mice administered etanercept, scR1antTNF-Fc (10 μg/kg), or 40-kDa PEG-scR1antTNF (10 μg/kg) (Fig. 9E). However, numbers of CD4+ Foxp3+ Tregs in scR1antTNF-Fc (10 μg/kg) or 40-kDa PEG-scR1antTNF (10 μg/kg)–treated mice were significantly higher than in saline-treated mice (Fig. 9F). Conversely, numbers of CD4+ Foxp3− Tconvs were significantly lower in scR1antTNF-Fc– and 40-kDa PEG-scR1antTNF–treated mice than in saline-treated mice. Similar states in these CD4+ T cell subpopulations were not observed in etanercept (1250 μg/kg)-treated mice. The Tregs/Tconvs ratio in the lymph nodes of scR1antTNF-Fc (10 μg/kg)– and 40-kDa PEG-scR1antTNF (10 μg/kg)–treated mice was also significantly greater than that in saline-treated mice. Therefore, the balance of immune cells in CIA mice was improved to normal conditions by the administration of the TNFR1 antagonist derivatives, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF. Two structurally optimized types of scR1antTNF, Fc-fusion and PEGylation, were effective at improving TNFR1 antagonist activity.

Figure 9.

Figure 9.

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PEGylated scR1antTNF suppresses arthritis in CIA mice as well as scR1antTNF-Fc. A, schematic structure modeling of 40-kDa PEG-scR1antTNF. Branched PEG, which has two 20-kDa PEG chains, was fused to scR1anTNF on an N-terminal amine group. Amino acid sequences, domain information, and molecular modification sites of 40-kDa PEG-scR1antTNF are described in Fig. S1B. B, saline (n = 8), etanercept (1250 μg/kg) (n = 8), scR1antTNF-Fc (10 μg/kg) (n = 8), and 40-kDa PEG-scR1antTNF (10 μg/kg) (n = 8) were administered i.p. twice a week to CIA mice from day 22 after immunization. Arthritis scores were evaluated for 3 weeks. Data are shown as the mean ± S.E.; *, p < 0.05 (one-way ANOVA with Tukey's multiple comparisons test). C, body weight was measured from day 22 to day 35. D, wrist joint swelling of a representative mouse in each group at day 35 is shown. E, the percentages of CD4+ T cells and CD8+ T cells in lymph node cells were analyzed in each treatment group on day 35 by FCM. F, the percentages of CD4+ Foxp3+ Tregs and CD4+ Foxp3− Tconvs in CD4+ T cells were analyzed at day 35. The ratio of Tregs/Tconvs was calculated from the results of individual mice. Data are shown as the mean ± S.D.; *, p < 0.05; **, p < 0.01 (one-way ANOVA with Tukey's multiple comparisons test).

Discussion

At present, anti-TNF drugs are used in the clinic to treat immunologic disorders. These molecules have diverse structure formats. Briefly, infliximab (23), adalimumab (24), and golimumab (25) are IgG derived from mouse or human structures. Etanercept has a TNFR2-human IgG-Fc domain fusion protein (26). Certolizumab pegol has a PEGylated Fab structure (27). These molecules have been developed to acquire advantages in terms of pharmaceutical effects or half-lives. Therapeutic drugs with artificial structures are likely to be widely used in the future.

We previously reported R1antTNF, a TNFα mutant with TNFR1-selective antagonistic activity (17). Almost all current biologics for the treatment of immunologic disorders target TNFα with the aim of suppressing TNF receptor signaling involved in inflammatory responses. However, our antagonist specifically targets TNFR1. R1antTNF effectively inhibits endogenous TNF for TNFR1 in a competitive manner, but it has a short t1/2 in vivo. Reduced stability or retention in plasma may require frequent administration. Therefore, methods to extend its stability in blood are required. From this point of view, we previously reported that a single-chain structure of R1antTNF enhanced its molecular stability and enabled easy molecular modification (20). The single-chain structure suppressed the instability or degradation of the homotrimeric structure of the cytokine or its derivatives. Furthermore, each N-terminal and C-terminal localized by a single-chain structure can contribute to an effective and uniform molecular modification (20). The 40-kDa PEGylation of scR1antTNF was easily constructed without a decrease in TNFR1 antagonistic activity.

In this study, we investigated the structural optimization of R1antTNF and created a scR1antTNF-Fc protein to further enhance stabilization in vivo. Fc fusion was expected to extend the plasma t1/2 based on a delay in its excretion because of its increased molecular weight and recycling via the FcRn receptor (28). An Expi293 expression system was applied to express the scR1antTNF-Fc protein. The protein was readily expressed in mammalian cells, even though the molecular weight was increased and the structure was complicated by Fc fusion. scR1antTNF-Fc (Vhss) and scR1antTNF-Fc (IL-2ss) have the same amino acid sequence because the signal sequence is removed during secretion. The expression system works with serum-free culture medium and therefore there is no concern about immunoglobulin contamination from fetal calf serum. The molecular characteristics of scR1antTNF-Fc such as in vitro receptor binding affinity, thermal stability, and TNF inhibitory activity were retained compared with scR1antTNF. Loss of TNFR1-selective antagonistic property by Fc fusion was not observed. The t1/2 of scR1antTNF-Fc in blood was substantially prolonged and longer than that of etanercept. The results indicated that the molecular modification of R1natTNF by a single-chain structure and Fc fusion dramatically improved its in vivo stability.

We confirmed the pharmaceutical effects of scR1antTNF-Fc and 40-kDa PEG-scR1antTNF in CIA mice. Treatment with scR1antTNF-Fc or 40-kDa PEG-scR1antTNF significantly suppressed arthritis in mice compared with saline. R1antTNF required daily administration such as twice a day to maintain blood levels, but these new TNFR1 antagonist derivatives showed anti-inflammatory effects by administration twice a week. Moreover, low-dose scR1antTNF-Fc or 40-kDa PEG-scR1antTNF had therapeutic effects in CIA mice similar to etanercept. Therefore, reduced amounts and dose frequency of TNFR1 antagonist derivatives effectively suppressed arthritis in CIA mice because scR1antTNF-Fc continued to inhibit TNF–TNFR1 interactions longer than R1antTNF.scR1antTNF-Fc (10 μg/kg) and 40-kDa PEG-scR1antTNF (10 μg/kg) improved the arthritis score, histopathology, and inflammatory cytokine production in CIA mice. These TNFR1 antagonist derivatives had the same arthritis-suppressing effect as etanercept. To investigate the characteristics of the TNFR1-selective inhibition mechanism of TNFR1 antagonist derivatives, we focused on immune cells involved in autoimmune pathology. We identified a subpopulation of CD4+ T cells in the lymph nodes that was different between CIA mice treated with TNFR1 antagonist derivatives (10 μg/kg) or etanercept (1250 μg/kg). TNFR1 antagonist derivatives and etanercept have different TNFα inhibitory mechanisms. Etanercept binds to TNFα and neutralizes its activity. TNFα transmits intracellular signaling through TNFR1 and TNFR2. Therefore, both TNFR1 and TNFR2 signaling pathways are blocked. However, TNFR1 antagonist derivatives such as scR1antTNF-Fc and 40-kDa PEG-scR1antTNF only bind to TNFR1 and block TNFR1 signaling selectively. Therefore, TNFR2 signaling is transmitted by endogenous TNFα. Recently, it was reported that remarkably high levels of TNFR2 were expressed in CD4+ Foxp3+ Tregs relative to CD4+ Foxp3− Tconvs (9–11). TNFR1 in both cells is barely expressed. Tregs, which are a subpopulation of CD4+ T cells, express the transcription factor Foxp3 and suppress excessive immune responses to maintain immune homeostasis (29, 30). Tregs prevent the development of autoimmune diseases such as rheumatoid arthritis (31), multiple sclerosis (32), inflammatory bowel diseases (33), psoriasis (34), type I diabetes (35), and systemic lupus erythematosus (36) by inhibiting effector T cell proliferation and cytokine production (37). We thought that TNFR1 antagonist derivatives might not block the proliferation or activation of Tregs by administration to CIA mice in contrast to etanercept. As a result of this mechanism, the TNFR1 antagonist derivative–treated group showed higher numbers of CD4+ Foxp3+ Tregs than the etanercept-treated group, and the ratio of CD4+ Foxp3+ Tregs/CD4+ Foxp3− Tconvs in the TNFR1 antagonist derivative–treated group was higher than that of the etanercept-treated group. This indicated that the TNFR1 antagonist derivatives improved the balance between Tregs and Tconvs to the normal state (Figs. 8C and 9F). In contrast to anti-TNF drugs for clinical use, we think that the TNFR1 antagonist derivatives scR1antTNF-Fc and 40-kDa PEG-scR1antTNF suppress inflammation via Tregs.

Furthermore, endogenous TNFα levels are increased during inflammation (38). Moreover, only scR1antTNF-Fc showed weak TNFR2 agonistic activity (Fig. 3D). It seems that the dimerization of scR1antTNF by Fc fusion is involved, although further studies are required to elucidate this TNFR2 agonist mechanism in detail. These conditions might contribute to the increase in Tregs.

Protein drugs have a therapeutic effect in immune diseases because they have a high target molecular specificity and a long t1/2 in the blood. However, TNFα blockers such as anti-TNF antibodies (e.g. infliximab and etanercept) are contraindicated for patients with demyelinating diseases such as multiple sclerosis, because they exacerbate disease. Therefore, Kv1.3 potassium channel inhibitors (dalazatide), JAK inhibitors (tofacitinib), and S1P receptor modulators (fingolimod), which are small molecule drugs, are expected to be a new category of therapeutic drugs for immune diseases (39–41). These new drugs inhibit T cell maturation, activation, and migration. We previously demonstrated that R1antTNF had a beneficial pharmacological effect in EAE mice, a multiple sclerosis model, by suppressing demyelination in the spinal cord and the proliferation of Th1 cells in lymph nodes (19). Therefore, we think that scR1antTNF-Fc and 40-kDa PEG-scR1antTNF will also be effective for the treatment of multiple sclerosis when using a low dose or low frequency of administration, because these R1antTNF derivatives have a prolonged t1/2 in the blood while retaining TNFR1 antagonist activity compared with R1antTNF. Moreover, because these TNFR1 antagonist derivatives have different modes of action from small molecule drugs, an additive and synergistic effect might be expected when used in combination. Further studies to determine whether scR1antTNF-Fc and 40-kDa PEG-scR1antTNF are effective in immune diseases including multiple sclerosis are required.

Here, we report a molecular modification to form a single-chain of the trimeric structure of R1antTNF followed by Fc-fusion and PEGylation for structural optimization and functionalization to overcome its low in vivo stability. These TNFR1 antagonist derivatives might be potential drug discovery seeds for the treatment of immunogenic disorders.

Experimental Procedures

Cell culture

LM cells were maintained in Eagle's minimum essential medium (Fujifilm Wako, Osaka, Japan) supplemented with 1% FBS and 1% antibiotic mixture (10,000 units/ml penicillin, 10 mg/ml streptomycin, and 25 μg/ml amphotericin B) (Fujifilm Wako). Ramos-Blue cells (human B lymphocyte cell line) were cultured in Iscove's modified Dulbecco's medium (Fujifilm Wako) supplemented with 10% FBS, 1% antibiotic mixture, and 100 μg/ml zeocin (Invivo Gen, San Diego, CA, USA). huTNFR2/mFas preadipocytes (TNFR1−/−/TNFR2−/− mouse preadipocytes expressing chimeric extracellular and transmembrane domains of human TNFR2 and the intracellular domain of mouse Fas), which were established previously (27), were cultured in DMEM (Fujifilm Wako) supplemented with 10% FBS, 1% antibiotic mixture, and 5 μg/ml blasticidin S HCl (Invitrogen).

Animal

BALB/c mice and DBA/1 mice were purchased from Oriental Yeast Co. Ltd. (Tokyo, Japan). All animal experiments were approved by the Kobe Gakuin University Experimental Animal Care and Use Committee (approval number A19–22; Kobe, Japan).

Molecular information of TNFR1 antagonists

The scR1antTNF·TNFR1 complex structure was modeled on those of R1antTNF (PDB ID 2E7A) and the lymphotoxin-α·TNFR1 complex (PDB ID 1TNR) using UCSF Chimera and MODELLER. Amino acid sequences, domain information, and molecular modification sites of scR1antTNF-Fc and 40-kDa PEG-scR1antTNF are provided in Fig. S1, A and B.

Cloning of scR1antTNF-Fc gene

The gene sequence of scR1antTNF was reported previously (20). The scR1antTNF gene (GenScript, Piscataway, NJ, USA) was fused to a signal peptide gene on the N-terminal by PCR. Two types of signal peptide derived from human IgG VH region (MGWSLILLFLVAVATGVHS) or human IL-2 (MYRMQLLSCIALSLALVTNS) were used. The resultant gene was inserted to a pCAG-Hyg human IgG-Fc fusion vector (Fujifilm Wako) to generate the scR1antTNF-Fc expression vector.

Expression and purification of TNF mutants

scR1antTNF-Fc protein was expressed using the Expi293 Expression System (Thermo Fisher Scientific) according to the manufacturer's protocol. In brief, the pCAG-scR1antTNF-Fc expression vector was transfected into Expi293F cells by a lipofection method using ExpiFectamine 293 reagent. 7 days later, cultured medium was collected by centrifugation. The scR1antTNF-Fc protein was recovered using KANEKA KanCapA (Fujifilm Wako) from the supernatant and eluted with 0.1 m glycine-HCl (pH 2.8). Recovered protein was further purified by size-exclusion chromatography using a HiLoad 16/600 Superdex 200 prep grade column (GE Healthcare, Piscataway, NJ, USA).

SDS-PAGE and immunoblotting

SDS-PAGE was performed using Tris-glycine-SDS buffer (pH 8.3), and proteins were detected by Coomassie Brilliant Blue stain. For Western blotting, proteins were transferred onto a PVDF membrane. After the blocking of unspecific binding proteins, scR1antTNF-Fc protein was probed with HRP-conjugated anti-human IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Visualization was performed by chemiluminescence with ECL Prime (GE Healthcare).

Binding kinetics analysis using SPR

Binding kinetics were analyzed by SPR (BIAcore X-100, GE Healthcare). Fc fusion proteins of human TNFR1 or TNFR2 (R&D Systems, Minneapolis, MN, USA) were immobilized on sensor chip CM5 by an amine coupling reaction. HBS-EP buffer (GE Healthcare) was used as running buffer. Proteins diluted with HBS-EP buffer were injected for 2 min at a flow rate of 30 μl/min, and dissociation was monitored for a further 2 min. The analysis was performed with single-cycle kinetics.

Thermal shift assay

scR1antTNF (250 μg/ml), etanercept (a commercially available TNF inhibitor, Enbrel, Pfizer, New York, NY, USA) (250 μg/ml) and scR1antTNF-Fcs (250 μg/ml) were serially diluted 2-fold with PBS (pH 7.4). The diluted proteins were mixed with SYPRO Orange (Protein Thermal Shift Dye, Applied Biosystems, Foster City, CA, USA). The mixtures were subsequently heat-denatured by raising the temperature from 25 to 90°C at a rate of 0.16°C/10 s, and fluorescence intensity was measured using a Step One Plus Real-Time PCR System (Applied Biosystems). To determine the Tm values, data were analyzed using Protein Thermal Shift Software v1.0 (Applied Biosystems).

In vitro bioactivity via TNFR1 or TNFR2

For cytotoxicity assays, LM cells (3 × 104 cells/well) were cultured with serially diluted TNFα or scR1antTNF-Fc at 37°C in 96-well plates. For the TNFα inhibition assay, LM cells (1 × 104 cells/well) were cultured with a serial dilution of scR1antTNF-Fc in the presence of mouse TNFα (5 ng/ml) (PeproTech, Rocky Hill, NJ, USA). 24 h later, cell viabilities in both assays were determined by methylene blue assay (42). For the NF-κB reporter assay, Ramos-Blue cells (3 × 105 cells/well) were cultured with serially diluted human TNFα, scR1antTNF or scR1antTNF-Fcs at 37°C. 24 h later, the supernatant was incubated with 1 mg/ml p-nitrophenyl phosphate buffer for 1 h at 37°C. NF-κB–inducible SEAP activity was calculated by measuring the absorbance at 405 nm. huTNFR2/mFas preadipocytes (1.5 × 104 cells/well) were cultured with serially diluted human TNFα or scR1antTNF-Fc. After incubation for 48 h at 37°C, cell viability was measured by a WST-8 colorimetric assay (Cell Counting Kit-8, Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's protocol.

Measurement of scR1antTNF-Fcs concentration in plasma

Female BALB/c mice (8 weeks old) were injected i.p. with 50 μg of scR1antTNF and scR1antTNF-Fcs, respectively. Saline and etanercept were injected as negative and positive controls, respectively. To investigate pharmacokinetics, blood was collected from the tail vein at 6, 24, 48, 72, 96, 168, and 192 h after injection. The concentration of each protein in plasma samples was measured using a human TNF ELISA kit (BD Biosciences, San Diego, CA, USA) or human IgG ELISA kit (Bethyl Laboratories, Montgomery, TX, USA). The half-life (t½) and AUC were calculated by moment analysis.

Induction of arthritis and administration of scR1antTNF-Fc

A mixture of bovine collagen type II (2 mg/ml, Chondrex, Redmond, WA, USA) and complete Freund's adjuvant (CFA, 3 mg/ml, Chondrex) were prepared according to a standard collagen-induced arthritis protocol by Chondrex, Inc. Then, 100 μl of the emulsion was subcutaneously immunized in DBA/1 mice (male, 6 weeks old) at the base of the tail. scR1antTNF-Fc, 40-kDa PEG-scR1antTNF, etanercept (positive control) and saline (negative control) were administered i.p. twice a week from day 22 after immunization.

Assessment of inflammation and body weight

The severity of arthritis was assessed by clinical score. Each paw was scored on a scale from 0 to 4 as follows: 0, normal paw; 1, mild but definite redness and swelling of the ankle or wrist or apparent redness and swelling limited to individual digits, regardless of the number of affected digits; 2, moderate redness and swelling of ankle or wrist; 3, severe redness and swelling of the entire paw including digits; and 4, maximally inflamed limb with involvement of multiple joints. The sum of the clinical scores of four paws was plotted as the degree of inflammation. Changes in body weight over the course of the experiment were measured.

Histological evaluation of arthritis

Mice were sacrificed at the end of the study period. The hind leg of each mouse was isolated and fixed in 10% neutral-buffered formalin. After decalcification in EDTA for 2 weeks, tissues were embedded in paraffin. The joint sections were placed onto microscopic slides and stained with H&E. According to a previous report (43), the degree of inflammation was determined based on H&E staining using the following observations: cell infiltration, synovitis, destruction of cartilage, and juxta-articular bone involvement. The grading was as follows: 0, normal (no inflammatory cells and normal appearance); 1, mild (a few inflammatory cells and mild thickening of the synovium with minor destruction of cartilage); 2, moderate (joint cavity partly filled with inflammatory cells and substantial thickening of the synovium with clear loss of cartilage); and 3, severe (joint cavity totally filled with inflammatory cells and severe thickening of the synovium with cartilage almost absent in the whole joint). TRAP staining was also performed with serial sections in accordance with a previous report (44). TRAP-positive multinucleated cells with ≥ single nuclei were counted as osteoclasts.

Quantification of cytokine level

Blood was collected using hematocrit tubes from the orbital sinus of anesthetized mice at day 42 after the administration of scR1antTNF-Fc. Then, plasma was prepared from the blood by centrifugation for 20 min at 15,000 × g. The concentration of mouse IL-1β in plasma was measured using a commercial ELISA kit (DuoSet, R&D Systems).

Cell isolation and flow cytometry (FCM) analysis

Axillary, brachial, and inguinal lymph nodes were isolated from mice sacrificed at the end of the study period. Single-cell suspensions were prepared by using a cell strainer (BD Biosciences). For the FCM analysis of lymph node cells, the following fluorochrome-conjugated antibodies were used: mouse CD4 (RM4-5), mouse CD8 (53-6.7) (BioLegend, San Diego, CA, USA), and mouse Foxp3 (FJK-16s) (Thermo Fisher Scientific) after mouse CD16/32 blocking. Cells were permeabilized and fixed using the Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer's instructions. Data were collected by CytoFLEX (Beckman Coulter, Brea, CA, USA) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

Statistical analysis

Means between groups were compared by one-way analysis of variance (ANOVA) followed by a secondary test (Tukey) to compare the groups two by two. p-Values of 0.05 or 0.01 were accepted as significant. All statistical analyses were carried out using GraphPad Prism version 6.0 for Windows Software (GraphPad Software, San Diego, CA, USA).

Data availability

All data except Protein Data Bank files (PDB IDs 2E7A and 1TNR) are contained within the article.

Supplementary Material

Supporting Information
supp_295_28_9379__index.html (1.2KB, html)

This article contains supporting information.

Author contributions—M. I. and S-i. T. conceptualization; M. I., H. K., Y. Tsutsumi, and S-i. T. resources; M. I. data curation; M. I. formal analysis; M. I., H. K., Y. Tsutsumi, and S-i. T. supervision; M. I. and S-i. T. funding acquisition; M. I. validation; M. I., Y. Tsuji, C. Y., S. E., Y. M., N. O., M. K., M. M., S. A., K. Y., and S-i. T. investigation; M. I. methodology; M. I. writing-original draft; M. I. and S-i. T. project administration; M. I. and S-i. T. writing-review and editing.

Funding and additional information—This work was supported by JSPS KAKENHI Grants JP18K14877, JP18H02699, and JP18K19567, and the Takeda Science Foundation and Mochida Memorial Foundation for Medical and Pharmaceutical Research.

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Abbreviations—The abbreviations used are:
TNF
tumor necrosis factor
TNFR
TNF receptor
Treg
regulatory T cell
CIA
collagen-induced arthritis
SPR
surface plasmon resonance
TSA
thermal shift assay
LM
mouse fibroblast
SEAP
secreted embryonic alkaline phosphatase
AUC
area under the curve
TRAP
tartrate-resistant acid phosphatase
Tconv
conventional T cell
FCM
flow cytometry
ANOVA
analysis of variance
RU
resonance unit.

References

  • 1. Smolen J. S., Aletaha D., Barton A., Burmester G. R., Emery P., Firestein G. S., Kavanaugh A., McInnes I. B., Solomon D. H., Strand V., and Yamamoto K. (2018) Rheumatoid arthritis. Nat. Rev. Dis. Primers 4, 18001 10.1038/nrdp.2018.1 [DOI] [PubMed] [Google Scholar]
  • 2. Smolen J. S., Aletaha D., and McInnes I. B. (2016) Rheumatoid arthritis. Lancet 388, 2023–2038 10.1016/S0140-6736(16)30173-8 [DOI] [PubMed] [Google Scholar]
  • 3. Gottenberg J. E., Brocq O., Perdriger A., Lassoued S., Berthelot J. M., Wendling D., Euller-Ziegler L., Soubrier M., Richez C., Fautrel B., Constantin A. L., Mariette X., Morel J., Gilson M., Cormier G., et al. (2016) Non-TNF-targeted biologic vs a second anti-TNF drug to treat rheumatoid arthritis in patients with insufficient response to a first anti-TNF drug: A randomized clinical trial. JAMA 316, 1172–1180 10.1001/jama.2016.13512 [DOI] [PubMed] [Google Scholar]
  • 4. Laas K., Peltomaa R., Kautiainen H., and Leirisalo-Repo M. (2008) Clinical impact of switching from infliximab to etanercept in patients with rheumatoid arthritis. Clin. Rheumatol. 27, 927–932 10.1007/s10067-008-0880-6 [DOI] [PubMed] [Google Scholar]
  • 5. van der Bijl A. E., Breedveld F. C., Antoni C. E., Kalden J. R., Kary S., Burmester G. R., Beckmann C., Unnebrink K., and Kupper H. (2008) An open-label pilot study of the effectiveness of adalimumab in patients with rheumatoid arthritis and previous infliximab treatment: Relationship to reasons for failure and anti-infliximab antibody status. Clin. Rheumatol. 27, 1021–1028 10.1007/s10067-008-0866-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Dong Y., Fischer R., Naudé P. J., Maier O., Nyakas C., Duffey M., Van der Zee E. A., Dekens D., Douwenga W., Herrmann A., Guenzi E., Kontermann R. E., Pfizenmaier K., and Eisel U. L. (2016) Essential protective role of tumor necrosis factor receptor 2 in neurodegeneration. Proc. Natl. Acad. Sci. U. S. A. 113, 12304–12309 10.1073/pnas.1605195113 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Chan F. K., Shisler J., Bixby J. G., Felices M., Zheng L., Appel M., Orenstein J., Moss B., and Lenardo M. J. (2003) A role for tumor necrosis factor receptor-2 and receptor-interacting protein in programmed necrosis and antiviral responses. J. Biol. Chem. 278, 51613–51621 10.1074/jbc.M305633200 [DOI] [PubMed] [Google Scholar]
  • 8. Kafrouni M. I., Brown G. R., and Thiele D. L. (2003) The role of TNF-TNFR2 interactions in generation of CTL responses and clearance of hepatic adenovirus infection. J. Leukoc. Biol. 74, 564–571 10.1189/jlb.0103035 [DOI] [PubMed] [Google Scholar]
  • 9. Chen X., Baümel M., Männel D. N., Howard O. M., and Oppenheim J. J. (2007) Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells. J. Immunol. 179, 154–161 10.4049/jimmunol.179.1.154 [DOI] [PubMed] [Google Scholar]
  • 10. Chen X., and Oppenheim J. J. (2010) TNF-alpha: An activator of CD4+FoxP3+TNFR2+ regulatory T cells. Curr. Dir. Autoimmun. 11, 119–134 10.1159/000289201 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Chen X., Subleski J. J., Kopf H., Howard O. M., Männel D. N., and Oppenheim J. J. (2008) Cutting edge: Expression of TNFR2 defines a maximally suppressive subset of mouse CD4+CD25+FoxP3+ T regulatory cells: Applicability to tumor-infiltrating T regulatory cells. J. Immunol. 180, 6467–6471 10.4049/jimmunol.180.10.6467 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Fischer R., Kontermann R., and Maier O. (2015) Targeting sTNF/TNFR1 signaling as a new therapeutic strategy. Antibodies 4, 48–70 10.3390/antib4010048 [DOI] [Google Scholar]
  • 13. Pegoretti V., Baron W., Laman J. D., and Eisel U. L. M. (2018) Selective modulation of TNF-TNFRs signaling: Insights for multiple sclerosis treatment. Front. Immunol. 9, 925 10.3389/fimmu.2018.00925 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Kodama S., Davis M., and Faustman D. L. (2005) The therapeutic potential of tumor necrosis factor for autoimmune disease: A mechanistically based hypothesis. Cell. Mol. Life Sci. 62, 1850–1862 10.1007/s00018-005-5022-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Ortí-Casañ N., Wu Y., Naudé P. J. W., De Deyn P. P., Zuhorn I. S., and Eisel U. L. M. (2019) Targeting TNFR2 as a novel therapeutic strategy for Alzheimer's disease. Front. Neurosci. 13, 49 10.3389/fnins.2019.00049 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Williams S. K., Fairless R., Maier O., Liermann P. C., Pichi K., Fischer R., Eisel U. L. M., Kontermann R., Herrmann A., Weksler B., Romero N., Couraud P. O., Pfizenmaier K., and Diem R. (2018) Anti-TNFR1 targeting in humanized mice ameliorates disease in a model of multiple sclerosis. Sci. Rep. 8, 13628 10.1038/s41598-018-31957-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Shibata H., Yoshioka Y., Ohkawa A., Minowa K., Mukai Y., Abe Y., Taniai M., Nomura T., Kayamuro H., Nabeshi H., Sugita T., Imai S., Nagano K., Yoshikawa T., Fujita T., et al. (2008) Creation and x-ray structure analysis of the tumor necrosis factor receptor-1-selective mutant of a tumor necrosis factor-α antagonist. J. Biol. Chem. 283, 998–1007 10.1074/jbc.M707933200 [DOI] [PubMed] [Google Scholar]
  • 18. Shibata H., Yoshioka Y., Abe Y., Ohkawa A., Nomura T., Minowa K., Mukai Y., Nakagawa S., Taniai M., Ohta T., Kamada H., Tsunoda S., and Tsutsumi Y. (2009) The treatment of established murine collagen-induced arthritis with a TNFR1-selective antagonistic mutant TNF. Biomaterials 30, 6638–6647 10.1016/j.biomaterials.2009.08.041 [DOI] [PubMed] [Google Scholar]
  • 19. Nomura T., Abe Y., Kamada H., Shibata H., Kayamuro H., Inoue M., Kawara T., Arita S., Furuya T., Yamashita T., Nagano K., Yoshikawa T., Yoshioka Y., Mukai Y., Nakagawa S., et al. (2011) Therapeutic effect of PEGylated TNFR1-selective antagonistic mutant TNF in experimental autoimmune encephalomyelitis mice. J. Controlled Release 149, 8–14 10.1016/j.jconrel.2009.12.015 [DOI] [PubMed] [Google Scholar]
  • 20. Inoue M., Ando D., Kamada H., Taki S., Niiyama M., Mukai Y., Tadokoro T., Maenaka K., Nakayama T., Kado Y., Inoue T., Tsutsumi Y., and Tsunoda S. I. (2017) A trimeric structural fusion of an antagonistic tumor necrosis factor-α mutant enhances molecular stability and enables facile modification. J. Biol. Chem. 292, 6438–6451 10.1074/jbc.M117.779686 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Dalton A. C., and Barton W. A. (2014) Over-expression of secreted proteins from mammalian cell lines. Protein Sci. 23, 517–525 10.1002/pro.2439 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Abe Y., Yoshikawa T., Kamada H., Shibata H., Nomura T., Minowa K., Kayamuro H., Katayama K., Miyoshi H., Mukai Y., Yoshioka Y., Nakagawa S., Tsunoda S., and Tsutsumi Y. (2008) Simple and highly sensitive assay system for TNFR2-mediated soluble- and transmembrane-TNF activity. J. Immunol. Methods 335, 71–78 10.1016/j.jim.2008.02.019 [DOI] [PubMed] [Google Scholar]
  • 23. Lipsky P. E., van der Heijde D. M., St. Clair E. W., Furst D. E., Breedveld F. C., Kalden J. R., Smolen J. S., Weisman M., Emery P., Feldmann M., Harriman G. R., and Maini R. N. (2000) Infliximab and methotrexate in the treatment of rheumatoid arthritis. Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant Therapy Study Group. N. Engl. J. Med. 343, 1594–1602 10.1056/NEJM200011303432202 [DOI] [PubMed] [Google Scholar]
  • 24. Wiens A., Correr C. J., Venson R., Otuki M. F., and Pontarolo R. (2010) A systematic review and meta-analysis of the efficacy and safety of adalimumab for treating rheumatoid arthritis. Rheumatol. Int. 30, 1063–1070 10.1007/s00296-009-1111-4 [DOI] [PubMed] [Google Scholar]
  • 25. Keystone E. C., Genovese M. C., Klareskog L., Hsia E. C., Hall S. T., Miranda P. C., Pazdur J., Bae S. C., Palmer W., Zrubek J., Wiekowski M., Visvanathan S., Wu Z., and Rahman M. U. (2009) Golimumab, a human antibody to tumour necrosis factor α given by monthly subcutaneous injections, in active rheumatoid arthritis despite methotrexate therapy: The GO-FORWARD Study. Ann. Rheum. Dis. 68, 789–796 10.1136/ard.2008.099010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Moreland L. W., Baumgartner S. W., Schiff M. H., Tindall E. A., Fleischmann R. M., Weaver A. L., Ettlinger R. E., Cohen S., Koopman W. J., Mohler K., Widmer M. B., and Blosch C. M. (1997) Treatment of rheumatoid arthritis with a recombinant human tumor necrosis factor receptor (p75)-Fc fusion protein. N. Engl. J. Med. 337, 141–147 10.1056/NEJM199707173370301 [DOI] [PubMed] [Google Scholar]
  • 27. Goel N., and Stephens S. (2010) Certolizumab pegol. MAbs 2, 137–147 10.4161/mabs.2.2.11271 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Roopenian D. C., and Akilesh S. (2007) FcRn: The neonatal Fc receptor comes of age. Nat. Rev. Immunol. 7, 715–725 10.1038/nri2155 [DOI] [PubMed] [Google Scholar]
  • 29. Sakaguchi S. (2005) Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat. Immunol. 6, 345–352 10.1038/ni1178 [DOI] [PubMed] [Google Scholar]
  • 30. Wing K., and Sakaguchi S. (2010) Regulatory T cells exert checks and balances on self tolerance and autoimmunity. Nat. Immunol. 11, 7–13 10.1038/ni.1818 [DOI] [PubMed] [Google Scholar]
  • 31. Biton J., Semerano L., Delavallée L., Lemeiter D., Laborie M., Grouard-Vogel G., Boissier M. C., and Bessis N. (2011) Interplay between TNF and regulatory T cells in a TNF-driven murine model of arthritis. J. Immunol. 186, 3899–3910 10.4049/jimmunol.1003372 [DOI] [PubMed] [Google Scholar]
  • 32. Kasagi S., Zhang P., Che L., Abbatiello B., Maruyama T., Nakatsukasa H., Zanvit P., Jin W., Konkel J. E., and Chen W. (2014) In vivo–generated antigen-specific regulatory T cells treat autoimmunity without compromising antibacterial immune response. Sci. Transl. Med. 6, 241ra278 10.1126/scitranslmed.3008895 [DOI] [PubMed] [Google Scholar]
  • 33. Mottet C., Uhlig H. H., and Powrie F. (2003) Cutting edge: Cure of colitis by CD4+CD25+ regulatory T cells. J. Immunol. 170, 3939–3943 10.4049/jimmunol.170.8.3939 [DOI] [PubMed] [Google Scholar]
  • 34. Wang H., Peters T., Sindrilaru A., Kess D., Oreshkova T., Yu X. Z., Seier A. M., Schreiber H., Wlaschek M., Blakytny R., Röhrbein J., Schulz G., Weiss J. M., and Scharffetter-Kochanek K. (2008) TGF-β-dependent suppressive function of Tregs requires wild-type levels of CD18 in a mouse model of psoriasis. J. Clin. Invest. 118, 2629–2639 10.1172/JCI34916 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Lindley S., Dayan C. M., Bishop A., Roep B. O., Peakman M., and Tree T. I. (2005) Defective suppressor function in CD4+CD25+ T-cells from patients with type 1 diabetes. Diabetes 54, 92–99 10.2337/diabetes.54.1.92 [DOI] [PubMed] [Google Scholar]
  • 36. La Paglia G. M. C., Leone M. C., Lepri G., Vagelli R., Valentini E., Alunno A., and Tani C. (2017) One year in review 2017: Systemic lupus erythematosus. Clin. Exp. Rheumatol. 35, 551–561 10.2337/diabetes.54.1.92 [DOI] [PubMed] [Google Scholar]
  • 37. Morgan M. E., Sutmuller R. P., Witteveen H. J., van Duivenvoorde L. M., Zanelli E., Melief C. J., Snijders A., Offringa R., de Vries R. R., and Toes R. E. (2003) CD25+ cell depletion hastens the onset of severe disease in collagen-induced arthritis. Arthritis Rheum. 48, 1452–1460 10.1002/art.11063 [DOI] [PubMed] [Google Scholar]
  • 38. Petrovic-Rackov L., and Pejnovic N. (2006) Clinical significance of IL-18, IL-15, IL-12 and TNF-α measurement in rheumatoid arthritis. Clin. Rheumatol. 25, 448–452 10.1007/s10067-005-0106-0 [DOI] [PubMed] [Google Scholar]
  • 39. Tarcha E. J., Olsen C. M., Probst P., Peckham D., Muñoz-Elias E. J., Kruger J. G., and Iadonato S. P. (2017) Safety and pharmacodynamics of dalazatide, a Kv1.3 channel inhibitor, in the treatment of plaque psoriasis: A randomized phase 1b trial. PLoS One 12, e0180762 10.1371/journal.pone.0180762 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. van Vollenhoven R. F., Fleischmann R., Cohen S., Lee E. B., García Meijide J. A., Wagner S., Forejtova S., Zwillich S. H., Gruben D., Koncz T., Wallenstein G. V., Krishnaswami S., Bradley J. D., Wilkinson B., and ORAL Standard Investigators. (2012) Tofacitinib or adalimumab versus placebo in rheumatoid arthritis. N. Engl. J. Med. 367, 508–519 10.1056/NEJMoa1112072 [DOI] [PubMed] [Google Scholar]
  • 41. Brinkmann V., Billich A., Baumruker T., Heining P., Schmouder R., Francis G., Aradhye S., and Burtin P. (2010) Fingolimod (FTY720): Discovery and development of an oral drug to treat multiple sclerosis. Nat. Rev. Drug Discov. 9, 883–897 10.1038/nrd3248 [DOI] [PubMed] [Google Scholar]
  • 42. Yamamoto Y., Tsutsumi Y., Yoshioka Y., Nishibata T., Kobayashi K., Okamoto T., Mukai Y., Shimizu T., Nakagawa S., Nagata S., and Mayumi T. (2003) Site-specific PEGylation of a lysine-deficient TNF-α with full bioactivity. Nat. Biotechnol. 21, 546–552 10.1038/nbt812 [DOI] [PubMed] [Google Scholar]
  • 43. Larsson E., Erlandsson Harris H., Larsson A., Mansson B., Saxne T., and Klareskog L. (2004) Corticosteroid treatment of experimental arthritis retards cartilage destruction as determined by histology and serum COMP. Rheumatology 43, 428–434 10.1093/rheumatology/keh073 [DOI] [PubMed] [Google Scholar]
  • 44. Yi H., Kim J., Jung H., Rim Y. A., Kim Y., Jung S. M., Park S. H., and Ju J. H. (2014) Induced production of anti-etanercept antibody in collagen-induced arthritis. Mol. Med. Rep. 9, 2301–2308 10.3892/mmr.2014.2127 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information
supp_295_28_9379__index.html (1.2KB, html)
supp_RA120.012723_158284_2_supp_527037_qc07jd.pdf (167KB, pdf)

Data Availability Statement

All data except Protein Data Bank files (PDB IDs 2E7A and 1TNR) are contained within the article.

Articles from The Journal of Biological Chemistry are provided here courtesy of American Society for Biochemistry and Molecular Biology

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