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Pharmacokinetic and Pharmacodynamic Characterisation of an Anti-Mouse TNF Receptor 1 Domain Antibody Formatted for In Vivo Half-Life Extension.

Goodall LJ, Ovecka M, Rycroft D, Friel SL, Sanderson A, Mistry P, Davies ML, Stoop AA - PLoS ONE (2015)

Bottom Line: Intriguingly, TNF-α signals through two receptors, TNFR1 and TNFR2, which have been associated with detrimental inflammatory and beneficial immune-regulatory processes, respectively.To investigate if selective TNFR1 inhibition might provide benefits over pan TNF-α inhibition, tools to investigate the potential impact of pharmacological intervention are needed.Surprisingly, the dAb did not compete with TNF-α for TNFR1-binding.

View Article: PubMed Central - PubMed

Affiliation: Biopharm Innovation Unit, Biopharm R&D, GlaxoSmithKline, Stevenage, United Kingdom.

ABSTRACT
Tumour Necrosis Factor-α (TNF-α) inhibition has been transformational in the treatment of patients with inflammatory disease, e.g. rheumatoid arthritis. Intriguingly, TNF-α signals through two receptors, TNFR1 and TNFR2, which have been associated with detrimental inflammatory and beneficial immune-regulatory processes, respectively. To investigate if selective TNFR1 inhibition might provide benefits over pan TNF-α inhibition, tools to investigate the potential impact of pharmacological intervention are needed. Receptor-deficient mice have been very insightful, but are not reversible and could distort receptor cross-talk, while inhibitory anti-TNFR1 monoclonal antibodies have a propensity to induce receptor agonism. Therefore, we set out to characterise a monovalent anti-TNFR1 domain antibody (dAb) formatted for in vivo use. The mouse TNFR1 antagonist (DMS5540) is a genetic fusion product of an anti-TNFR1 dAb with an albumin-binding dAb (AlbudAb). It bound mouse TNFR1, but not human TNFR1, and was an antagonist of TNF-α-mediated cytotoxicity in a L929 cell assay. Surprisingly, the dAb did not compete with TNF-α for TNFR1-binding. This was supported by additional data showing the anti-TNFR1 epitope mapped to a single residue in the first domain of TNFR1. Pharmacokinetic studies of DMS5540 in mice over three doses (0.1, 1.0 and 10 mg/kg) confirmed extended in vivo half-life, mediated by the AlbudAb, and demonstrated non-linear clearance of DMS5540. Target engagement was further confirmed by dose-dependent increases in total soluble TNFR1 levels. Functional in vivo activity was demonstrated in a mouse challenge study, where DMS5540 provided dose-dependent inhibition of serum IL-6 increases in response to bolus mouse TNF-α injections. Hence, DMS5540 is a potent mouse TNFR1 antagonist with in vivo pharmacokinetic and pharmacodynamic properties compatible with use in pre-clinical disease models and could provide a useful tool to dissect the individual contributions of TNFR1 and TNFR2 in homeostasis and disease.

No MeSH data available.


Related in: MedlinePlus

DMS5540 PD determined by protection provided to IL-6 serum increases after in vivo TNF-α challenge.Eight mice per dose group were injected with DMS5540, DMS5538 (Dummy control) or nothing at the indicated dose in mg/kg. Four hours later all mice indicated received a bolus injection of mouse TNF-α (100 ng/mouse) and 2 hours later serum samples were taken to determine IL-6 levels. (A) Boxplot for IL-6 serum level (pg/ml) for each dose group. Boxplot description: the horizontal line is the median and the diamond is the mean. The upper and lower ends of the box are located at the upper quartile (Q3) and lower quartile (Q1) respectively. The whiskers show the minimum observation before 1.5 x IQR below the box and the maximum observation before 1.5 x IQR above the box. Observations beyond the error bars are shown as individual data points. (B) Individual mouse IL-6 serum levels (pg/ml) for all 8 mice in each dose group.
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pone.0137065.g005: DMS5540 PD determined by protection provided to IL-6 serum increases after in vivo TNF-α challenge.Eight mice per dose group were injected with DMS5540, DMS5538 (Dummy control) or nothing at the indicated dose in mg/kg. Four hours later all mice indicated received a bolus injection of mouse TNF-α (100 ng/mouse) and 2 hours later serum samples were taken to determine IL-6 levels. (A) Boxplot for IL-6 serum level (pg/ml) for each dose group. Boxplot description: the horizontal line is the median and the diamond is the mean. The upper and lower ends of the box are located at the upper quartile (Q3) and lower quartile (Q1) respectively. The whiskers show the minimum observation before 1.5 x IQR below the box and the maximum observation before 1.5 x IQR above the box. Observations beyond the error bars are shown as individual data points. (B) Individual mouse IL-6 serum levels (pg/ml) for all 8 mice in each dose group.

Mentions: Finally, we set out to investigate the pharmacodynamic properties of DMS5540 in an in vivo challenge study. The challenge model consisted of DMS5540-dosed mice receiving an intravenous bolus injection of mouse TNF-α and determining the effects on serum IL-6 levels. The chosen TNF-α dose (100 ng/25g mouse) generated a robust IL-6 response without leading to any visible discomfort to the mice or any behavioural changes that would indicate ethical concerns for the study to progress. In these studies, eight mice per dose group were first injected intravenously with i) a range of DMS5540 concentrations (0.1–3.0 mg/kg), ii) the negative control dAb (DMS5538) (3 mg/kg) or iii) no dAb. Four hours later, all these mice received mouse TNF-α (100ng/mouse). Serum samples were taken 2 hrs after the TNF-α challenge and the serum levels of IL-6 were determined. The IL-6 response data are summarised in boxplots for each dose group in Fig 5A and shown for each individual mouse in Fig 5B. To determine whether the observed differences were significant, a statistical analysis was done in which the adjusted (least square) geometric means for the different treatment groups were compared and the 95% Confidence Intervals (CI) were calculated (Table 3). From the 0.3 mg/kg dose of DMS5540 upwards, the observed decrease in IL-6 response becomes significant compared to the TNF-α only group (p = 0.02). For the 1 mg/kg dose group, IL-6 levels were inhibited by >95%, which was highly significant compared to either the TNF-α only or negative control dAb (DMS5538) group, p = 0.0001 or p = 0.001, respectively. The differences observed for the 3 mg/kg dose group continued to be significant compared to both control groups. Hence, DMS5540 demonstrated the ability to inhibit TNF-α-mediated signalling effects in vivo and could therefore be an appropriate tool reagent to investigate the effects of selective, pharmacological inhibition of mouse TNFR1 in pre-clinical disease models.


Pharmacokinetic and Pharmacodynamic Characterisation of an Anti-Mouse TNF Receptor 1 Domain Antibody Formatted for In Vivo Half-Life Extension.

Goodall LJ, Ovecka M, Rycroft D, Friel SL, Sanderson A, Mistry P, Davies ML, Stoop AA - PLoS ONE (2015)

DMS5540 PD determined by protection provided to IL-6 serum increases after in vivo TNF-α challenge.Eight mice per dose group were injected with DMS5540, DMS5538 (Dummy control) or nothing at the indicated dose in mg/kg. Four hours later all mice indicated received a bolus injection of mouse TNF-α (100 ng/mouse) and 2 hours later serum samples were taken to determine IL-6 levels. (A) Boxplot for IL-6 serum level (pg/ml) for each dose group. Boxplot description: the horizontal line is the median and the diamond is the mean. The upper and lower ends of the box are located at the upper quartile (Q3) and lower quartile (Q1) respectively. The whiskers show the minimum observation before 1.5 x IQR below the box and the maximum observation before 1.5 x IQR above the box. Observations beyond the error bars are shown as individual data points. (B) Individual mouse IL-6 serum levels (pg/ml) for all 8 mice in each dose group.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4564187&req=5

pone.0137065.g005: DMS5540 PD determined by protection provided to IL-6 serum increases after in vivo TNF-α challenge.Eight mice per dose group were injected with DMS5540, DMS5538 (Dummy control) or nothing at the indicated dose in mg/kg. Four hours later all mice indicated received a bolus injection of mouse TNF-α (100 ng/mouse) and 2 hours later serum samples were taken to determine IL-6 levels. (A) Boxplot for IL-6 serum level (pg/ml) for each dose group. Boxplot description: the horizontal line is the median and the diamond is the mean. The upper and lower ends of the box are located at the upper quartile (Q3) and lower quartile (Q1) respectively. The whiskers show the minimum observation before 1.5 x IQR below the box and the maximum observation before 1.5 x IQR above the box. Observations beyond the error bars are shown as individual data points. (B) Individual mouse IL-6 serum levels (pg/ml) for all 8 mice in each dose group.
Mentions: Finally, we set out to investigate the pharmacodynamic properties of DMS5540 in an in vivo challenge study. The challenge model consisted of DMS5540-dosed mice receiving an intravenous bolus injection of mouse TNF-α and determining the effects on serum IL-6 levels. The chosen TNF-α dose (100 ng/25g mouse) generated a robust IL-6 response without leading to any visible discomfort to the mice or any behavioural changes that would indicate ethical concerns for the study to progress. In these studies, eight mice per dose group were first injected intravenously with i) a range of DMS5540 concentrations (0.1–3.0 mg/kg), ii) the negative control dAb (DMS5538) (3 mg/kg) or iii) no dAb. Four hours later, all these mice received mouse TNF-α (100ng/mouse). Serum samples were taken 2 hrs after the TNF-α challenge and the serum levels of IL-6 were determined. The IL-6 response data are summarised in boxplots for each dose group in Fig 5A and shown for each individual mouse in Fig 5B. To determine whether the observed differences were significant, a statistical analysis was done in which the adjusted (least square) geometric means for the different treatment groups were compared and the 95% Confidence Intervals (CI) were calculated (Table 3). From the 0.3 mg/kg dose of DMS5540 upwards, the observed decrease in IL-6 response becomes significant compared to the TNF-α only group (p = 0.02). For the 1 mg/kg dose group, IL-6 levels were inhibited by >95%, which was highly significant compared to either the TNF-α only or negative control dAb (DMS5538) group, p = 0.0001 or p = 0.001, respectively. The differences observed for the 3 mg/kg dose group continued to be significant compared to both control groups. Hence, DMS5540 demonstrated the ability to inhibit TNF-α-mediated signalling effects in vivo and could therefore be an appropriate tool reagent to investigate the effects of selective, pharmacological inhibition of mouse TNFR1 in pre-clinical disease models.

Bottom Line: Intriguingly, TNF-α signals through two receptors, TNFR1 and TNFR2, which have been associated with detrimental inflammatory and beneficial immune-regulatory processes, respectively.To investigate if selective TNFR1 inhibition might provide benefits over pan TNF-α inhibition, tools to investigate the potential impact of pharmacological intervention are needed.Surprisingly, the dAb did not compete with TNF-α for TNFR1-binding.

View Article: PubMed Central - PubMed

Affiliation: Biopharm Innovation Unit, Biopharm R&D, GlaxoSmithKline, Stevenage, United Kingdom.

ABSTRACT
Tumour Necrosis Factor-α (TNF-α) inhibition has been transformational in the treatment of patients with inflammatory disease, e.g. rheumatoid arthritis. Intriguingly, TNF-α signals through two receptors, TNFR1 and TNFR2, which have been associated with detrimental inflammatory and beneficial immune-regulatory processes, respectively. To investigate if selective TNFR1 inhibition might provide benefits over pan TNF-α inhibition, tools to investigate the potential impact of pharmacological intervention are needed. Receptor-deficient mice have been very insightful, but are not reversible and could distort receptor cross-talk, while inhibitory anti-TNFR1 monoclonal antibodies have a propensity to induce receptor agonism. Therefore, we set out to characterise a monovalent anti-TNFR1 domain antibody (dAb) formatted for in vivo use. The mouse TNFR1 antagonist (DMS5540) is a genetic fusion product of an anti-TNFR1 dAb with an albumin-binding dAb (AlbudAb). It bound mouse TNFR1, but not human TNFR1, and was an antagonist of TNF-α-mediated cytotoxicity in a L929 cell assay. Surprisingly, the dAb did not compete with TNF-α for TNFR1-binding. This was supported by additional data showing the anti-TNFR1 epitope mapped to a single residue in the first domain of TNFR1. Pharmacokinetic studies of DMS5540 in mice over three doses (0.1, 1.0 and 10 mg/kg) confirmed extended in vivo half-life, mediated by the AlbudAb, and demonstrated non-linear clearance of DMS5540. Target engagement was further confirmed by dose-dependent increases in total soluble TNFR1 levels. Functional in vivo activity was demonstrated in a mouse challenge study, where DMS5540 provided dose-dependent inhibition of serum IL-6 increases in response to bolus mouse TNF-α injections. Hence, DMS5540 is a potent mouse TNFR1 antagonist with in vivo pharmacokinetic and pharmacodynamic properties compatible with use in pre-clinical disease models and could provide a useful tool to dissect the individual contributions of TNFR1 and TNFR2 in homeostasis and disease.

No MeSH data available.


Related in: MedlinePlus