The TNFR1 Antagonist Atrosimab Is Therapeutic in Mouse Models of Acute and Chronic Inflammation


doi: 10.3389/fimmu.2021.705485.


eCollection 2021.

Affiliations

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Fabian Richter et al.


Front Immunol.


.

Abstract

Therapeutics that block tumor necrosis factor (TNF), and thus activation of TNF receptor 1 (TNFR1) and TNFR2, are clinically used to treat inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease and psoriasis. However, TNFR1 and TNFR2 work antithetically to balance immune responses involved in inflammatory diseases. In particular, TNFR1 promotes inflammation and tissue degeneration, whereas TNFR2 contributes to immune modulation and tissue regeneration. We, therefore, have developed the monovalent antagonistic anti-TNFR1 antibody derivative Atrosimab to selectively block TNFR1 signaling, while leaving TNFR2 signaling unaffected. Here, we describe that Atrosimab is highly stable at different storage temperatures and demonstrate its therapeutic efficacy in mouse models of acute and chronic inflammation, including experimental arthritis, non-alcoholic steatohepatitis (NASH) and experimental autoimmune encephalomyelitis (EAE). Our data support the hypothesis that it is sufficient to block TNFR1 signaling, while leaving immune modulatory and regenerative responses via TNFR2 intact, to induce therapeutic effects. Collectively, we demonstrate the therapeutic potential of the human TNFR1 antagonist Atrosimab for treatment of chronic inflammatory diseases.


Keywords:

EAE; TNF; TNFR1; arthritis; inflammatory diseases; multiple sclerosis.

Conflict of interest statement

Author M-AD, SH and AH were employed by company Baliopharm. FR, AH, KP and REK are named inventors on patents and patent applications covering the Atrosimab technology. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from Baliopharm. The funder was involved in the design of the experimental arthritis study.

Figures


Figure 1



Figure 1

Biochemical and functional characterization of Atrosimab. (A) Atrosimab was analyzed by size exclusion chromatography (SEC) using a TSKgel SuperSW mAb high resolution column. (B) Atrosimab was resolved by SDS-PAGE under reducing (R) and non-reducing (NR) conditions and protein was visualized using Coomassie Brilliant Blue staining (M = markers). (C, D) Thermal stability was analyzed by (C) dynamic light scattering and (D) dynamic scanning calorimetry. (E, F) Atrosimab stored for the indicated times in 50% human serum at 37°C was added to HT1080 cells followed by addition of (E) 0.01 nM TNF or (F) without TNF. Atrosab stored at 5°C was used as a positive control. After 16-20 hours incubation at 37°C, 5% CO2, the supernatants were analyzed for secreted IL-8 by ELISA. Mean ± SD, n=3, Atrosab vs Atrosimab 1 day: *p < 0.05.


Figure 2



Figure 2

Comparative pharmacokinetic analysis of Atrosimab and ATROSAB. Atrosimab or Atrosab were injected (A, B) intravenously (i.v.) or (C, D) subcutaneously (s.c.) at doses of 1.0 mg/kg or 30.0 mg/kg body weight into (A, C) C57BL/6 wildtype (WT) or (B, D) C57BL/6 huTNFR1-k/i mice. Serum samples, collected at the indicated time points, were analyzed for remaining circulating protein by ELISA. Shown are the mean ± SD of three mice.


Figure 3



Figure 3

Atrosimab blocks acute TNF-induced inflammation in vivo.
(A–C) C57BL/6 huTNFR1-k/i mice received i.v. injections of TNF (0.3 mg/kg), Atrosimab or FcΔab (30 mg/kg). (A) Body weight was determined and (B, C) blood samples were collected before treatment (0 h) and 1 h, 6 h, 24 h and 72 h after the injections. (B) IL-6 and (C) CRP levels in the serum were determined by ELISA. (D–F) C57BL/6 huTNFR1-k/i mice received i.v. injections of PBS, Atrosimab (30 mg/kg body weight) or FcΔab (30 mg/kg). TNF (0.3 mg/kg) was injected 30 minutes thereafter to induce acute inflammatory responses. (D) Body weight was determined and (E, F) blood samples were collected before treatment (0 h) and 1 h, 6 h, 24 h and 72 h after TNF injection. (E) IL-6 and (F) CRP levels in the serum were determined by ELISA. Individual values were excluded using the ROUT method of outlier identification or because they were below the detection limit (Q = 1%) or because they were below the detection limit of the ELISA. Mean ± SD, n=6. (A, C): **p < 0.01; (D): *p < 0.05 – TNF + PBS vs TNF + Atrosimab, #p < 0.05 – TNF + PBS vs TNF + FcΔab; (E, F): ns, not significant, **p < 0.01.


Figure 4



Figure 4

Atrosimab prevents development of arthritic disease. Tg197hTNFR1KI transgenic mice were used as a model of experimental arthritis. Treatment (s.c.) with saline, the anti-TNF therapeutics Etanercept or Infliximab (20 mg/kg each), or different doses of Atrosimab was started at 4 weeks of age and repeated twice weekly. Arthritis score (A) and weight (B) were documented weekly. (C, D) Histopathological analysis of the joints was performed at an age of 15 weeks and compared to 4-week-old non-treated mice. Shown are (C) representative pictures and (D) quantification of the histological (H) score. Mean ± SD, n=8, *p < 0.05, ***p < 0.001.


Figure 5



Figure 5

Atrosimab is therapeutic for arthritic disease. Tg197hTNFR1KI transgenic mice were treated at an age of 9 weeks with saline, the anti-TNF therapeutics Infliximab or Certolizumab pegol (15 mg/kg each), or different doses of Atrosimab by twice weekly s.c. injections. Arthritis score (A) and weight (B) were documented weekly. (C, D) Histopathological analysis of the joints was performed at an age of 15 weeks and compared to 9-week-old non-treated mice. Shown are representative pictures and quantification of the histological (H) score. Mean ± SD, n=8, *p < 0.05, ***/###p < 0.001.


Figure 6



Figure 6

Atrosimab alleviated fibrosis, steatosis and liver injury in a model of NAFLD. huTNFR1-k/i mice were fed a high-fat diet for 21 weeks and then treated with saline, Atrosimab and Atrosab for 8 weeks. Liver pathology was analyzed by quantifying (A, B) liver fibrosis, (C, D) steatosis and (E, F) liver injury. (A, B) Liver fibrosis was quantified using Sirius Red staining, (C, D) liver steatosis using Oil Red O staining to identify triglyceride content and (E, F) liver injury by detection of caspase-3 activity. Shown are representative pictures (A, C, E) and quantification of the histological analyses (B, D, F). a: scale bars = 100 µm, c,e: scale bars = 50 µm. Mean ± SEM, (B): n=9 saline, n=8 Atrosab, n=7 Atrosimab; (D): n=9 saline, n=6 Atrosab, n=5 Atrosimab, (F): n=5 each: *p < 0.05, **p < 0.01.


Figure 7



Figure 7

Atrosimab ameliorates disease in a model of multiple sclerosis. huTNFR1-k/i mice were immunized with MOG35-55 peptide to induce EAE. Mice were injected i.p. on days 1, 4, 8 and 12 of manifest disease with either 30 mg/kg or 60 mg/kg Atrosimab, or 30 mg/kg FcΔab. Both dosages of Atrosimab led to an immediate and sustained improvement in (A) motor symptoms, reflected by the (B) cumulative EAE score, as well as a reduction in (C) disease-associated weight loss, also supported by (D) the cumulative weight loss. Two of the major pathological hallmarks of EAE, demyelination and axonal degeneration were investigated histopathologically. (E–G) Luxol fast blue (LFB) staining revealed, in comparison to FcΔab treated animals, significantly reduced demyelination following treatment with 30 mg/kg Atrosimab. (H–J) Treatment with 30 mg/kg Atrosimab also lead to a reduction in the number of damaged axons indicated by APP accumulation. Representative images are shown from Fc control (E, H) and Atrosimab-treated (F, I) mice. e,f,h,i: scale bars = 50 µm. a-d: Mean ± SEM, n=6; g,j: n=6 per group: *p<0.05, **p<0.01.

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Dies ist ein automatisch übersetzter Artikel. Er kann nur einer groben Orientierung dienen. Das Original gibt es hier: psoriasis

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