Structure-guided Optimization of the Interleukin-6 Trans-signaling Antagonist sgp130

Tenhumberg, Stephanie; Waetzig, Georg H.; Chalaris, Athena; Rabe, Björn; Seegert, Dirk; Scheller, Jürgen; Rose-John, Stefan; Grötzinger, Joachim

doi:10.1074/jbc.m803694200

Cited by 61 publications

(73 citation statements)

References 43 publications

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“…Production and Characterization of sgp130Fc-dNGsgp130Fc and sgp130Fc-dNG were produced in stable clonal CHO-K1 production cell lines in Ham's F-12 medium supplemented with 5% IgG-stripped FBS (both from PAA Laboratories) in roller bottles (2,125 cm 2 ; Greiner Bio-One, Frickenhausen, Germany) and purified by protein A affinity chromatography as described previously (18,29). Protein integrity was analyzed by native PAGE and SDS-PAGE and subsequent silver staining (29).…”

Section: Methodsmentioning

confidence: 99%

“…Constructs and Tools-A codon and structurally optimized version of human sgp130Fc has been described before (28,29). An analogous codon-optimized variant sgp130Fc-dNG was synthesized by GENEART (Regensburg, Germany), in which all nine occupied N-glycosylation sites described by Moritz et al (21) were mutated from Asn to Gln.…”

Section: Methodsmentioning

confidence: 99%

“…Their proliferation upon stimulation with Hyper-IL-6, a covalent fusion protein of human IL-6 and sIL-6R (32), or IL-6⅐sIL-6R as well as the dose-dependent inhibition of this process by sgp130Fc (and lack of inhibition by sgp130Fc-dNG) were measured by the cell titer colorimetric assay (Promega) as described previously (29).…”

Section: Methodsmentioning

confidence: 99%

“…Protein integrity was analyzed by native PAGE and SDS-PAGE and subsequent silver staining (29). "Control-Fc" was constructed by fusing the IgG1-Fc contained in sgp130Fc and sgp130Fc-dNG to an Ig signal peptide from pSecTag2 (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

“…The apparent molecular weight of sgp130Fc and the control protein fetuin were analyzed by SDS-PAGE with 7.5 and 10% gels, respectively, and subsequent silver staining (29).…”

Section: Methodsmentioning

confidence: 99%

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N-Linked Glycosylation Is Essential for the Stability but Not the Signaling Function of the Interleukin-6 Signal Transducer Glycoprotein 130

Waetzig

Chalaris

Rosenstiel³

et al. 2010

Journal of Biological Chemistry

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N-Linked glycosylation is an important determinant of protein structure and function. The interleukin-6 signal transducer glycoprotein 130 (gp130) is a common co-receptor for cytokines of the interleukin (IL)-6 family and is N-glycosylated at 9 of 11 potential sites. Whereas N-glycosylation of the extracellular domains D1-D3 of gp130 has been shown to be dispensable for binding of the gp130 ligand IL-6 and its cognate receptor in vitro, the role of the N-linked glycans on domains D4 and D6 is still unclear. We have mutated the asparagines of all nine functional N-glycosylation sites of gp130 to glutamine and systematically analyzed the consequences of deleted N-glycosylation (dNG) in both cellular gp130 and in a soluble gp130-IgG1-Fc fusion protein (sgp130Fc). Our results show that sgp130Fc-dNG is inherently unstable and degrades rapidly under conditions that do not harm wild-type sgp130Fc. Consistently, the bulk of cellular gp130-dNG is not transported to the plasma membrane but is degraded in the proteasome. However, the small quantities of gp130-dNG, which do reach the cell surface, are still able to activate the key gp130 signaling target signal transducer and activator of transcription-3 (STAT3) upon binding of the agonistic complex of IL-6 and soluble IL-6 receptor. In conclusion, N-linked glycosylation is required for the stability but not the signal-transducing function of gp130.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…The apparent molecular weight of sgp130Fc and the control protein fetuin were analyzed by SDS-PAGE with 7.5 and 10% gels, respectively, and subsequent silver staining (29).…”

Section: Methodsmentioning

confidence: 99%

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N-Linked Glycosylation Is Essential for the Stability but Not the Signaling Function of the Interleukin-6 Signal Transducer Glycoprotein 130

Waetzig

Chalaris

Rosenstiel³

et al. 2010

Journal of Biological Chemistry

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Effect of Induction Therapy With Olamkicept vs Placebo on Clinical Response in Patients With Active Ulcerative Colitis

Zhang

Chen

Wang

et al. 2023

JAMA

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ImportanceOlamkicept, a soluble gp130-Fc-fusion-protein, selectively inhibits interleukin 6 (IL-6) trans-signaling by binding the soluble IL-6 receptor/IL-6 complex. It has anti-inflammatory activities in inflammatory murine models without immune suppression.ObjectiveTo assess the effect of olamkicept as induction therapy in patients with active ulcerative colitis.Design, Setting, and ParticipantsRandomized, double-blind, placebo-controlled phase 2 trial of olamkicept in 91 adults with active ulcerative colitis (full Mayo score ≥5, rectal bleeding score ≥1, endoscopy score ≥2) and an inadequate response to conventional therapy. The study was conducted at 22 clinical study sites in East Asia. Patients were recruited beginning in February 2018. Final follow-up occurred in December 2020.InterventionsEligible patients were randomized 1:1:1 to receive a biweekly intravenous infusion of olamkicept 600 mg (n = 30) or 300 mg (n = 31) or placebo (n = 30) for 12 weeks.Main Outcomes and MeasuresThe primary end point was clinical response at week 12 (defined as ≥3 and ≥30% decrease from baseline total Mayo score; range, 0-12 [worst] with ≥1 decrease and ≤1 in rectal bleeding [range, 0-3 {worst}]). There were 25 secondary efficacy outcomes, including clinical remission and mucosal healing at week 12.ResultsNinety-one patients (mean age, 41 years; 25 women [27.5%]) were randomized; 79 (86.8%) completed the trial. At week 12, more patients receiving olamkicept 600 mg (17/29 [58.6%]) or 300 mg (13/30 [43.3%]) achieved clinical response than placebo (10/29 [34.5%]), with adjusted difference vs placebo of 26.6% (90% CI, 6.2% to 47.1%; P = .03) for 600 mg and 8.3% (90% CI, −12.6% to 29.1%; P = .52) for 300 mg. Among patients randomized to receive 600 mg olamkicept, 16 of 25 secondary outcomes were statistically significant compared with placebo. Among patients randomized to receive 300 mg, 6 of 25 secondary outcomes were statistically significant compared with placebo. Treatment-related adverse events occurred in 53.3% (16/30) of patients receiving 600 mg olamkicept, 58.1% (18/31) receiving 300 mg olamkicept, and 50% (15/30) receiving placebo. The most common drug-related adverse events were bilirubin presence in the urine, hyperuricemia, and increased aspartate aminotransferase levels, and all were more common in the olamkicept groups compared with placebo.Conclusions and RelevanceAmong patients with active ulcerative colitis, biweekly infusion of olamkicept 600 mg, but not 300 mg, resulted in a greater likelihood of clinical response at 12 weeks compared with placebo. Further research is needed for replication and to assess longer-term efficacy and safety.Trial RegistrationClinicalTrials.gov Identifier: NCT03235752

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Receptor‐Fc and Ligand Traps as High‐Affinity Biological Blockers: Development and Clinical Applications

Economides

Stahl

2013

Fusion Protein Technologies for Biopharmaceuticals

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Structure-guided Optimization of the Interleukin-6 Trans-signaling Antagonist sgp130

Cited by 61 publications

References 43 publications

N-Linked Glycosylation Is Essential for the Stability but Not the Signaling Function of the Interleukin-6 Signal Transducer Glycoprotein 130

N-Linked Glycosylation Is Essential for the Stability but Not the Signaling Function of the Interleukin-6 Signal Transducer Glycoprotein 130

Effect of Induction Therapy With Olamkicept vs Placebo on Clinical Response in Patients With Active Ulcerative Colitis

Receptor‐Fc and Ligand Traps as High‐Affinity Biological Blockers: Development and Clinical Applications

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