Andrew Nesbitt

Mechanism of action of certolizumab pegol (CDP870): In vitro comparison with other anti-tumor necrosis factor α agents

Background: Inhibitors of tumor necrosis factor &agr; (TNF&agr;) have demonstrated significant efficacy in chronic inflammatory diseases, including Crohns disease (CD). To further elucidate the mechanisms of action of these agents, we compared the anti‐TNF&agr; agents certolizumab pegol, infliximab, adalimumab, and etanercept in several in vitro systems. Methods: The ability of each anti‐TNF&agr; agent to neutralize soluble and membrane‐bound TNF&agr;; mediate cytotoxicity, affect apoptosis of activated human peripheral blood lymphocytes and monocytes; induce degranulation of human peripheral blood granulocytes, and modulate lipopolysaccharide (LPS)‐induced interleukin (IL)‐1&bgr; production by human monocytes was measured in vitro. Results: All 4 agents neutralized soluble TNF&agr; and bound to and neutralized membrane TNF&agr;. Infliximab and adalimumab were comparable in their ability to mediate complement‐dependent cytotoxicity and antibody‐dependent cell‐mediated cytotoxicity, and to increase the proportion of cells undergoing apoptosis and the level of granulocyte degranulation. Etanercept generally mediated these effects to a lesser degree, while certolizumab pegol gave similar results to the control reagents. LPS‐induced IL‐1&bgr; production was inhibited by certolizumab pegol, infliximab, and adalimumab, but only partially inhibited by etanercept. Conclusions: In contrast to the other anti‐TNF&agr; agents tested, certolizumab pegol did not mediate increased levels of apoptosis in any of the in vitro assays used, suggesting that these mechanisms are not essential for the efficacy of anti‐TNF&agr; agents in CD. As certolizumab pegol, infliximab, and adalimumab, but not etanercept, almost completely inhibited LPS‐induced IL‐1&bgr; release from monocytes, inhibition of cytokine production may be important for efficacy of anti‐TNF&agr; agents in CD.

Use of biofluorescence imaging to compare the distribution of certolizumab pegol, adalimumab, and infliximab in the inflamed paws of mice with collagen-induced arthritis

Exposure to a drug at the site of inflammation may be an important consideration for the effective treatment of inflammatory disorders such as rheumatoid arthritis (RA). The purpose of this in vivo study was to identify a methodology to enable effective quantification of antibody-type reagents in normal and inflamed tissue by investigating the distribution of the tumor necrosis factor-alpha (TNF-alpha) inhibitors, certolizumab pegol, adalimumab, and infliximab, in healthy and inflamed murine tissue using a novel non-invasive biofluorescence method. Certolizumab pegol, adalimumab, and infliximab were labeled with the low molecular weight dye alexa680. The agents were administered intravenously at a dose of 2mg/kg in naïve DBA/1 mice and in DBA/1 mice with ongoing collagen-induced arthritis. Concentrations of the TNF inhibitors in the hind paws were measured using a Xenogen IVIS200 biofluorescence imager at multiple time points up to 26h post-administration. In 2 independent experiments, the distribution of certolizumab pegol was compared with that of adalimumab and infliximab. Certolizumab pegol, adalimumab, and infliximab all distributed more effectively into inflamed tissue than non-inflamed tissue in this animal model of arthritis. However, the ratio of penetration of certolizumab pegol into inflamed arthritic paws compared with normal tissue was greater than that observed with adalimumab and infliximab. Furthermore, the duration of exposure in the inflamed versus normal tissue was more prolonged for certolizumab pegol than for both adalimumab and infliximab, and the accumulation of certolizumab pegol in diseased tissue was more responsive to the severity of inflammation when compared with adalimumab and infliximab. It is probable that these features of certolizumab pegol are conferred on the molecule by PEGylation. It is important to assess exposure to drug at the site of inflammation, because distinct structural features of certain agents may affect efficacy, tolerability, rapidity and/or sustainability of effect. The novel non-invasive biofluorescence method used in this study is an effective tool for comparing tissue penetration of therapeutic agents.

A PEGylated Fab’ Fragment against Tumor Necrosis Factor for the Treatment of Crohn Disease

Antibodies, having a high specificity for their particular target, are increasingly being used as therapeutic agents with functions including agonist, antagonist, and targeted drug delivery. The use of many biologic therapies, including antibody fragments, is generally limited by their rapid clearance from plasma. A commonly used approach to extend exposure to biologic therapies is the attachment of polyethylene glycol.Tumor necrosis factor (TNF)-α is a multifunctional cytokine involved in the regulation of immune responses. Elevated levels of TNFα are found in a wide range of diseases, including the chronic inflammatory conditions rheumatoid arthritis, psoriasis, and Crohn disease (CD). Anti-TNFα antibodies have proved highly efficacious in the treatment of these conditions. In addition, they have proved invaluable for investigating the role of TNFα in disease etiology.Based on evidence showing that neutralizing antibodies to TNFα were effective in animal models of CD, anti-TNFα antibody treatments were assessed in clinical trials. Interestingly, the anti-TNFα antibody etanercept proved ineffective at achieving remission in active CD despite potently neutralizing soluble TNFα. This indicated that an additional mode of action is also involved in the efficacy of the anti-TNFα agents adalimumab, certolizumab pegol, and infliximab in CD; one suggestion was apoptosis. However, etanercept, like adalimumab and infliximab, can induce apoptosis. Furthermore, certolizumab pegol (which has demonstrated efficacy in CD) does not cause complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, apoptosis, or necrosis of neutrophils, all measured in vitro. These functional differences observed with certolizumab pegol stem from its unique structure that does not include the crystallizable fragment (Fc) portion present in the other anti-TNFα agents, and the way in which it signals through membrane TNF.It is well established that bacteria are a major part of the inflammatory process in CD. The property identified that reflected the efficacies of the anti-TNFα agents etanercept, adalimumab, certolizumab pegol, and infliximab in CD was the ability to inhibit the cytokine production by monocytes that is induced by bacterial lipopolysaccharide. It may therefore be the case that this mode of action is important for efficacy in CD.

Certolizumab pegol does not bind the neonatal Fc receptor (FcRn): Consequences for FcRn-mediated in vitro transcytosis and ex vivo human placental transfer

Antibodies to tumor necrosis factor (anti-TNF) are used to treat inflammatory diseases, which often affect women of childbearing age. The active transfer of these antibodies across the placenta by binding of the Fc-region to the neonatal Fc receptor (FcRn) may result in adverse fetal or neonatal effects. In contrast to other anti-TNFs, certolizumab pegol lacks an Fc-region. The objective of this study was to determine whether the structure of certolizumab pegol limits active placental transfer. Binding affinities of certolizumab pegol, infliximab, adalimumab and etanercept to human FcRn and FcRn-mediated transcytosis were determined using in vitro assays. Human placentas were perfused ex vivo to measure transfer of certolizumab pegol and positive control anti-D IgG from the maternal to fetal circulation. FcRn binding affinity (KD) was 132nM, 225nM and 1500nM for infliximab, adalimumab and etanercept, respectively. There was no measurable certolizumab pegol binding affinity, similar to that of the negative control. FcRn-mediated transcytosis across a cell layer (mean±SD; n=3) was 249.6±25.0 (infliximab), 159.0±20.2 (adalimumab) and 81.3±13.1ng/mL (etanercept). Certolizumab pegol transcytosis (3.2±3.4ng/mL) was less than the negative control antibody (5.9±4.6ng/mL). No measurable transfer of certolizumab pegol from the maternal to the fetal circulation was observed in 5 out of 6 placentas that demonstrated positive-control IgG transport in the ex vivo perfusion model. Together these results support the hypothesis that the unique structure of certolizumab pegol limits its transfer through the placenta to the fetus and may be responsible for previously reported differences in transfer of other anti-TNFs from mother to fetus.

FRI0162 Investigation into the binding affinity of certolizumab pegol to fcrn and functional consequences for fcrn-mediated transcytosis: comparison to infliximab, adalimumab and etanercept

Background Certolizumab pegol (CZP) is a PEGylated, Fc-free anti-TNF that lacks the Fc portion found in monoclonal antibodies. Infliximab (IFX) and adalimumab (ADA) are both antibodies, while etanercept (ETA) is a receptor fusion protein, and all three of these anti-TNFs have an IgG1 Fc. In mothers treated with CZP, it has been reported that lower levels of CZP, compared to ADA or IFX, are transferred to the neonate.1 It has been suggested this transfer differential may be due to the one-way active transport of antibodies across the placenta thought to be mediated by the neonatal Fc receptor (FcRn). However, anti-TNF binding to FcRn, and FcRn-mediated transcytosis across a cell layer, have not been studied. Objectives To quantify binding of the anti-TNFs CZP, IFX, ADA and ETA to FcRn and to measure FcRn-mediated transcytosis of these agents across a cell layer. Methods A Biacore™ assay was used to determine the binding of CZP, ADA and IFX to human FcRn. Anti-TNFs were passed over an FcRn-coated chip for 5 minutes at a range of concentrations from 21-670nM to determine the on-binding rate; a buffer at pH 6.0 was used to allow optimum binding. The off-rate was followed for a further 5 minutes by running buffer alone over the chip. MDCK II cells transfected with human FcRn were used to measure FcRn-mediated transcytosis across a cell layer using a pH 5.9 buffer on the apical side and pH 7.2 on the basolateral side. The anti-TNFs and the control antibody (P146), which possessed a Fc modified to prevent binding to FcRn, were biotinylated to allow visualization. The amount of each anti-TNF transcytosed across the cell layer over 4 hours was measured by MSD assay. Results IFX (132nM) and ADA (225nM) had relatively high binding affinity to FcRn while the binding affinity of ETA to FcRn was approximately 5 to 10-fold lower (1500nM), similar to previously reported results.2 In contrast, CZP did not bind to the FcRn with any measurable affinity. The levels of transcytosis seen with IFX and ADA were 249.6ng/mL and 159.5ng/mL, respectively (mean of 3 experiments), over 4 hours. Transcytosis of ETA (81.3ng/mL) was lower than that of ADA and IFX. In contrast, the level of CZP transcytosis was significantly lower, at 3.2ng/mL, than that observed with the other anti-TNFs tested. The control antibody P146 also showed a low level of transfer at 5.9ng/mL. Since neither the control antibody nor CZP bind to FcRn, the levels detected are probably due to a low level of non-specific leakage across the cell layer. Conclusions This is the first report to quantify the binding of anti-TNFs to FcRn and their FcRn-mediated transcytosis across a cell layer. CZP does not have an Fc and thus did not bind to FcRn. Moreover, no FcRn-mediated CZP transcytosis was detected. In contrast, ADA and IFX had a relatively high binding affinity to FcRn and were actively transcytosed across the cell layer. ETA showed lower binding affinity to FcRn and subsequent transcytosis, compared to IFX and ADA, but FcRn-mediated transport could still be measured. These results explain the previously observed active transport of anti-TNFs across the placenta seen in patients treated with IFX and ADA, whereas only low levels were observed with CZP.1 References Mahadevan U. Clin Gastroenterol Hepatol 2012 [epub ahead of print]; 2. Suzuki T. J Immunol 2010;184(4):1968-1976. Acknowledgements The authors acknowledge Costello Medical Consulting for writing and editorial assistance which was funded by UCB Pharma. Disclosure of Interest T. Baker Employee of: UCB Pharma, L. Kevorkian Employee of: UCB Pharma, A. Nesbitt Shareholder of: UCB Pharma, Employee of: UCB Pharma

Certolizumab pegol: a PEGylated anti-tumour necrosis factor alpha biological agent

Tumour necrosis factor (TNF)α is a proinflammatory cytokine involved in systemic inflammation that mediates chronic inflammatory diseases such as rheumatoid arthritis (RA), Crohn’s disease (CD) and psoriasis. Recognition of TNFα as a primary mediator of inflammatory disease has driven the development of monoclonal antibodies (mAbs) against TNFα as potential novel therapies for these disorders. Certolizumab pegol is a novel, polyethylene glycol (PEG)-conjugated, humanised, antigen-binding fragment (Fab’) of an anti-TNFα mAb that does not mediate apoptosis or neutrophil degranula- tion. Preclinical studies have shown excellent bioavailability, with preferential distribution and retention in inflamed tissue, which could be due to the low diffusion rate of PEGylated molecules and/or the lack of an Fc, which prevents FcRn-mediated transport. Pharmacokinetics are linear and predictable. Certolizumab pegol is a potentially valuable new treatment option for several inflammatory diseases. It has shown promising efficacy and tolerability results in Phase II and III trials for RA, CD and psoriasis.

Mapping the certolizumab pegol epitope on TNF and comparison with infliximab, adalimumab and etanercept.

Certolizumab pegol (CZP) has a different mode of action from the other anti-TNFs, infliximab (IFX), adalimumab (ADA) and etanercept (ETA), that may be due both to its structure and how it signals through membrane TNF-α (mTNF-α). Different effects of this signalling could relate to the exact epitopes to which the anti-TNFs bind. We investigated the binding epitope for CZP and how it differs from the other anti-TNFs.

Reverse signalling of membrane TNF in human natural killer cells: a comparison of the effect of certolizumab pegol and other anti-TNF agents

Differences have been seen among the anti-TNFs in mediating reverse signalling of membrane TNF-α (mTNF-α). Natural killer (NK) cells express high levels of mTNF-α and may be involved in rheumatoid arthritis pathogenesis. We examined the effect of certolizumab pegol (CZP) and the other anti-TNFs, adalimumab (ADA), etanercept (ETA) and infliximab (IFX), on cellular activities of NK cells.

Tu1876 The Effects of Anti-TNF Agents on the Expansion of T Helper-Type 17 (TH17) Cells Driven by Lipopolysaccharide-Stimulated Monocytes

Patients and methods Monocytes and CD4+ cells were purified, by positive and negative selection, from peripheral blood mononuclear cells of healthy volunteers. CD4+CD45RO+ memory T cells were enriched from the CD4+ cell fraction by positive selection. A 1:1 ratio of monocytes and memory T cells was co-cultured for 7 days with CD3/CD28 Human T-Activator Dynabeads and 1 μg/mL LPS. Cells were cultured with and without 10 μg/mL anti-TNF agent. Subsequently, CD4+ cells were stained for intracellular Interferon g (INFg) and IL-17A, and analyzed by flow cytometry. IL-17A and IL-17F secretion was determined by ELISA.