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

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.

Tumour necrosis factor receptor I blockade shows that TNF-dependent and TNF-independent mechanisms synergise in TNF receptor associated periodic syndrome.

TNF receptor associated periodic syndrome (TRAPS) is an autoinflammatory disease involving recurrent episodes of fever and inflammation. It is associated with autosomal dominant mutations in TNF receptor superfamily 1A gene localised to exons encoding the ectodomain of the p55 TNF receptor, TNF receptor-1 (TNFR1). The aim of this study was to investigate the role of cell surface TNFR1 in TRAPS, and the contribution of TNF-dependent and TNF-independent mechanisms to the production of cytokines. HEK-293 and SK-HEP-1 cell lines were stably transfected with WT or TRAPS-associated variants of human TNF receptor superfamily 1A gene. An anti-TNFR1 single domain antibody (dAb), and an anti-TNFR1 mAb, bound to cell surface WT and variant TNFR1s. In HEK-293 cells transfected with death domain-inactivated (R347A) TNFR1, and in SK-HEP-1 cells transfected with normal (full-length) TNFR1, cytokine production stimulated in the absence of exogenous TNF by the presence of certain TNFR1 variants was not inhibited by the anti-TNFR1 dAb. In SK-Hep-1 cells, specific TRAPS mutations increased the level of cytokine response to TNF, compared to WT, and this augmented cytokine production was suppressed by the anti-TNFR1 dAb. Thus, TRAPS-associated variants of TNFR1 enhance cytokine production by a TNF-independent mechanism and by sensitising cells to a TNF-dependent stimulation. The TNF-dependent mechanism requires cell surface expression of TNFR1, as this is blocked by TNFR1-specific dAb.

Selective tumor necrosis factor receptor I blockade is antiinflammatory and reveals immunoregulatory role of tumor necrosis factor receptor II in collagen-induced arthritis.

OBJECTIVE: Tumor necrosis factor (TNF) signals via 2 receptors, TNFR type I (TNFRI) and TNFRII, with distinct cellular distribution and signaling functions. In rheumatoid arthritis (RA), the net effect of TNFR signaling favors inflammatory responses while inhibiting the activity of regulatory T cells. TNFRII signaling has been shown to promote Treg cell function. To assess the relative contributions of TNFRI and TNFRII signaling to inflammatory and regulatory responses in vivo, we compared the effect of TNF blockade, hence TNFRI/II, versus TNFRI alone in collagen-induced arthritis (CIA) as a model of RA. METHODS: Mice with established arthritis were treated for 10 days with anti-mouse TNFRI domain antibody (dAb; DMS5540), an isotype control dAb (DMS5538), or murine TNFRII genetically fused with mouse IgG1 Fc domain (mTNFRII-Fc) beginning on the day of arthritis onset, and disease progression was monitored. Systemic cytokine concentrations and numbers of T cell subsets in lymph nodes and spleens were measured, and intrinsic Treg cell function was determined by ex vivo suppression assays. RESULTS: Progression of CIA was suppressed similarly by TNFRI (DMS5540) and TNFRI/II (mTNFRII-Fc) blockade. However, blockade of TNFRI/II led to increased effector T cell activity, which was not observed after selective TNFRI blockade, suggesting an immunoregulatory role of TNFRII. In support of this, TNFRI blockade, but not TNFRI/II blockade, expanded and activated Treg cells. Furthermore, a dramatic increase in expression of the Treg cell signature genes FoxP3 and TNFRII was observed in joints undergoing remission, which supports the notion that these molecules have a physiologic role in the resolution of inflammation. CONCLUSION: We propose that a therapeutic strategy that targets TNFRI while sparing TNFRII has the potential to both inhibit inflammation and promote Treg cell activity, which might be superior to TNF blockade.

TNFR2 increases the sensitivity of ligand-induced activation of the p38 MAPK and NF-κB pathways and signals TRAF2 protein degradation in macrophages.

Tumour necrosis factor (p55 or p60) receptor (TNFR) 1 is the major receptor that activates pro-inflammatory signalling and induces gene expression in response to TNF. Consensus is lacking for the function of (p75 or p80) TNFR2 but experiments in mice have suggested neuro-, cardio- and osteo-protective and anti-inflammatory roles. It has been shown in various cell types to be specifically required for the induction of TNFR-associated factor-2 (TRAF2) degradation and activation of the alternative nuclear factor (NF)-kappaB pathway, and to contribute to the activation of mitogen-activated protein kinases (MAPK) and the classical NF-kappaB pathway. We have investigated the signalling functions of TNFR2 in primary human and murine macrophages. We find that in these cells TNF induces TRAF2 degradation, and this is blocked in TNFR2(-/-) macrophages. TRAF2 has been previously reported to be required for TNF-induced activation of p38 MAPK. However, TRAF2 degradation does not inhibit TNF-induced tolerance of p38 MAPK activation. Neither TNF, nor lipopolysaccharide treatment, induced activation of the alternative NF-kappaB pathway in macrophages. Activation by TNF of the p38 MAPK and NF-kappaB pathways was blocked in TNFR1(-/-) macrophages. In contrast, although TNFR2(-/-) macrophages displayed robust p38 MAPK activation and IkappaBα degradation at high concentrations of TNF, at lower doses the concentration dependence of signalling was weakened by an order of magnitude. Our results suggest that, in addition to inducing TRAF2 protein degradation, TNFR2 also plays a crucial auxiliary role to TNFR1 in sensitising macrophages for the ligand-induced activation of the p38 MAPK and classical NF-kappaB pro-inflammatory signalling pathways.

Selective blockade of tumor necrosis factor receptor I inhibits proinflammatory cytokine and chemokine production in human rheumatoid arthritis synovial membrane cell cultures.

OBJECTIVE: To determine whether selective blockade of tumor necrosis factor receptor I (TNFRI) affects spontaneous proinflammatory cytokine and chemokine production in ex vivo-cultured human rheumatoid arthritis synovial membrane mononuclear cells (MNCs) and to compare this response to that of TNF ligand blockade using etanercept. METHODS: A bispecific, single variable-domain antibody (anti-TNFRI moiety plus an albumin binding moiety [TNFRI-AlbudAb]) was used to selectively block TNFRI. Inhibition of TNFα-mediated responses in cell lines expressing TNFRI/II confirmed TNFRI-AlbudAb potency, human rhabdomyosarcoma cell line KYM-1D4 cytotoxicity, and human umbilical vein endothelial cell (HUVEC) vascular cell adhesion molecule 1 (VCAM-1) upregulation. Eighteen RA synovial membrane MNC suspensions were cultured for 2 days or 5 days, either alone or in the presence of TNFRI-AlbudAb, control-AlbudAb, or etanercept. Proinflammatory cytokines and chemokines in culture supernatants were measured by enzyme-linked immunosorbent assays. A mixed-effects statistical analysis model was used to assess the extent of TNFRI selective blockade, where the results were expressed as the percentage change with 95% confidence intervals (95% CIs). RESULTS: TNFRI-AlbudAb inhibited TNFα-induced KYM-1D4 cell cytotoxicity (50% inhibition concentration [IC50 ] 4 nM) and HUVEC VCAM-1 up-regulation (IC50 12 nM) in a dose-dependent manner. In ex vivo-cultured RA synovial membrane MNCs, selective blockade of TNFRI inhibited the production of proinflammatory cytokines and chemokines to levels similar to those obtained with TNF ligand blockade, without inducing cellular toxicity. Changes in cytokine levels were as follows: -23.5% (95% CI -12.4, -33.2 [P = 0.004]) for granulocyte-macrophage colony-stimulating factor, -33.4% (95% CI -20.6, -44.2 [P ≤ 0.0001]) for interleukin-10 (IL-10), -17.6% (95% CI 3.2, -34.2 [P = 0.0880]) for IL-1ß, and -19.0% (95% CI -3.4, -32.1 [P = 0.0207]) for IL-6. Changes in chemokine levels were as follows: -34.2% (-14.4, -49.4 [P = 0.0030]) for IL-8, -56.6% (-30.7, -72.9 [P = 0.0011]) for RANTES, and -24.9% (2, -44.8 [P = 0.0656]) for monocyte chemotactic protein 1. CONCLUSION: In ex vivo-cultured RA synovial membrane MNCs, although a limited role of TNFRII cannot be ruled out, TNFRI signaling was found to be the dominant pathway leading to proinflammatory cytokine and chemokine production. Thus, selective blockade of TNFRI could potentially be therapeutically beneficial over TNF ligand blockade by retaining the beneficial TNFRII signaling.

Analysis of an engineered plasma kallikrein inhibitor and its effect on contact activation.

Engineering of protein-protein interactions is used to enhance the affinity or specificity of proteins, such as antibodies or protease inhibitors, for their targets. However, fully diversifying all residues in a protein-protein interface is often unfeasible. Therefore, we limited our phage library for the serine protease inhibitor ecotin by restricting it to only tetranomial diversity and then targeted all 20 amino acid residues involved in protein recognition. This resulted in a high-affinity and highly specific plasma kallikrein inhibitor, ecotin-Pkal. To validate this approach we dissected the energetic contributions of each wild type (wt) or mutated surface loop to the binding of either plasma kallikrein (PKal) or membrane-type serine protease 1. The analysis demonstrated that a mutation in one loop has opposing effects depending on the sequence of surrounding loops. This finding stresses the cooperative nature of loop-loop interactions and justifies targeting multiple loops with a limited diversity. In contrast to ecotin wt, the specific loop combination of ecotin-Pkal discriminates the subtle structural differences between the active enzymes, PKal and Factor XIIa, and their respective zymogen forms. We used ecotin-Pkal to specifically inhibit contact activation of human plasma at the level mediated by plasma kallikrein.

Engineering of a macromolecular scaffold to develop specific protease inhibitors.

The specific inhibition of serine proteases, which are crucial switches in many physiologically important processes, is of value both for basic research and for therapeutic applications. Ecotin, a potent macromolecular inhibitor of serine proteases of the S1A family, presents an attractive scaffold to engineer specific protease inhibitors because of its large inhibitor-protease interface. Using synthetic shuffling in combination with a restricted tetranomial diversity, we created ecotin libraries that are mutated at all 20 amino acid residues in the binding interface. The efficacy of these libraries was demonstrated against the serine protease plasma kallikrein (Pkal). Competitive phage display selection yielded a Pkal inhibitor with an apparent dissociation equilibrium constant (K(i)*) of 11 pM, whereas K(i)* values for related proteases (such as Factor Xa (FXa), Factor XIa (FXIa), urokinase-type plasminogen activator (uPA), thrombin, and membrane-type serine protease 1 (MT-SP1)) were four to seven orders of magnitude higher. The adaptability of the scaffold was demonstrated by the isolation of inhibitors to two additional serine proteases, MT-SP1/matriptase and Factor XIIa.