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- MATERIALS AND METHODS
Background: Inhibitors of tumor necrosis factor α (TNFα) have demonstrated significant efficacy in chronic inflammatory diseases, including Crohn's disease (CD). To further elucidate the mechanisms of action of these agents, we compared the anti-TNFα agents certolizumab pegol, infliximab, adalimumab, and etanercept in several in vitro systems.
Methods: The ability of each anti-TNFα agent to neutralize soluble and membrane-bound TNFα; 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)-1β production by human monocytes was measured in vitro.
Results: All 4 agents neutralized soluble TNFα and bound to and neutralized membrane TNFα. 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-1β production was inhibited by certolizumab pegol, infliximab, and adalimumab, but only partially inhibited by etanercept.
Conclusions: In contrast to the other anti-TNFα 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-TNFα agents in CD. As certolizumab pegol, infliximab, and adalimumab, but not etanercept, almost completely inhibited LPS-induced IL-1β release from monocytes, inhibition of cytokine production may be important for efficacy of anti-TNFα agents in CD.
Tumor necrosis factor α (TNFα) is a pluripotent cytokine that is present as both soluble (17 kDa) and membrane-bound (26 kDa) forms and mediates a diverse range of biologic effects. TNFα monomers associate to form a trimeric complex that is capable of binding to both TNFα receptor (TNFR)-1 and TNFR-2. Although the precise function of TNFα in health and disease is unclear, elevated levels of TNFα have been found in patients experiencing inflammatory diseases, and clinical data on the efficacy of anti-TNFα agents suggest that it plays a key role in the pathogenesis of a number of inflammatory diseases such as Crohn's disease (CD) and rheumatoid arthritis (RA). However, differences between the clinical profiles of the currently marketed anti-TNFα agents suggest that neutralization of soluble TNFα may not be the only mechanism of action of these agents. The availability of a number of anti-TNFα therapies with different structures and in vitro properties offers the opportunity to investigate the potential role of these properties in clinical efficacy.
Certolizumab pegol (Cimzia™; UCB, Belgium) is a PEGylated Fab′ fragment of a humanized monoclonal antibody that binds and neutralizes human TNFα. The pharmaco-kinetic properties of Fab′ in vivo are usually poor. However, attachment of a 40 kDa polyethylene glycol (PEG) moiety to the Fab′ fragment markedly increases the half-life of certolizumab to a value comparable with that of a whole antibody product. The Fab′ fragment was engineered with a single free-cysteine residue in the hinge region, which enables site-specific attachment of PEG without affecting the ability of the Fab′ fragment to bind and neutralize TNFα.
The unique structure of certolizumab pegol differs from that of other anti-TNFα agents that have been tested for the treatment of CD, i.e., infliximab (a mouse–human chimeric whole antibody) (Remicade™; Centocor, Horsham, PA, USA), adalimumab (a recombinant human whole antibody) (Humira; Abbott Laboratories, Abbott Park, IL, USA), and etanercept (a TNFR2–human IgFc fusion protein) (Enbrel™; Amgen, Thousand Oaks, CA, USA). The latter 3 agents are all based on the human IgG1 Fc (which has the capability of fixing complement and binding to Fc receptors) and therefore have the potential for Fc-mediated effects. Infliximab has demonstrated efficacy in the management of symptoms in patients with active CD.1 In contrast, etanercept failed to show efficacy in a double-blind, placebo-controlled Phase II study.2 A number of reasons have been proposed to explain these observations including variations in the penetration of the diseased tissue among the anti-TNF agents or differences in stability of the immune complexes with TNFα but, importantly, these data suggest that neutralization of soluble TNFα (sTNFα) is not sufficient to induce and maintain remission in active CD. Other mechanisms of action proposed to be important for the efficacy of anti-TNFα agents in CD are: induction of apoptosis of both monocytes3, 4 and T cells5, 6; neutralization of membrane TNFα (mTNFα)5; antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)7; and reverse signaling via mTNFα.8
To investigate whether the structure of certolizumab pegol could result in a different mechanism of action from that of infliximab, adalimumab, and etanercept, we assessed the 4 agents in a series of in vitro studies: neutralization of sTNFα and mTNFα; induction of ADCC, CDC, and apoptosis; degranulation of neutrophils; and inhibition of lipopolysaccharide (LPS)-induced cytokine production.
- Top of page
- MATERIALS AND METHODS
Therapeutic agents targeting TNFα were evaluated in clinical trials in a number of inflammatory disease settings including CD, RA, ankylosing spondylitis, and psoriasis.10 However, the diverse range of biologic effects mediated by TNFα has hampered efforts to define the mechanisms of action of these agents.
Recently released positive clinical data from the Phase III PRECiSE studies of certolizumab pegol in CD11, 12 have provided an opportunity to reassess available data from studies of infliximab, adalimumab, and etanercept. Certolizumab pegol, infliximab, and adalimumab appear to be similar in terms of induction and maintenance of response and remission, as reported in the similarly designed PRECiSE 2,11 ACCENT I,13 and CHARM14 trials. Infliximab is also effective in ulcerative colitis and for fistulizing disease and mucosal healing in CD; studies have not yet been published for certolizumab pegol in these indications.
The structure of certolizumab pegol differs significantly from the other agents that have undergone clinical evaluation in CD. Therefore, it is becoming possible for the first time to begin to determine which facets of an anti-TNFα agent may be required for clinical efficacy in CD and which may be unnecessary and potentially undesirable.
The studies described here were undertaken to compare the 4 agents in a range of in vitro experimental systems. Most of these studies used the anti-TNFα agents at a concentration of up to 100 μg/mL to reflect the concentration of these drugs found in the blood of patients, which are all in the μg/mL range. As expected, all the agents potently neutralized the bioactivity of sTNFα. These data are consistent with published data with infliximab and etanercept.5 Although neutralization of sTNFα is likely to play an important role in the efficacy profile of anti-TNFα agents across a range of inflammatory diseases, these data suggest that this mechanism alone does not predict clinical efficacy in CD, because of the apparent failure of etanercept in Phase II clinical trials for CD.2
TNFα expressed on the membrane of cells appears to be capable of mediating a range of effects, and mTNFα can bind and stimulate TNFα receptors on other cells.15 In the studies described here, certolizumab pegol bound to and neutralized effects mediated by mTNFα. It has been suggested previously that etanercept does not bind to mTNFα to the same extent as infliximab5, 16, 17 and the experiments described in this report generally support these observations. However, etanercept was clearly capable of some degree of binding to mTNFα, as suggested by its ability to neutralize mTNFα-mediated stimulation of a reporter cell line and to mediate CDC and ADCC (Figs. 4A,B). With the exception of CDC, etanercept generally appeared less potent in these assays than infliximab and adalimumab.
Certolizumab pegol did not mediate either CDC or ADCC (Figs. 4A,B), as would be predicted from the absence of an Fc region in the molecule. Both infliximab and adalimumab effectively bound and neutralized mTNFα (Figs. 2, 3) and mediated CDC and ADCC (Figs. 4A,B), consistent with data from other experimental systems.7, 16 It is possible that the different results obtained with etanercept in the experimental systems used in these studies and previously published data may relate to differences in the proportions of monomeric and trimeric TNFα expressed by the respective cell lines used. Based on these data, binding to monomeric TNFα cannot therefore be excluded as a potential requirement for efficacy in CD. Etanercept is also different from the other reagents in that it neutralizes lymphotoxin in addition to TNFα,16 although it is unclear if this fact is significant with regard to the lack of efficacy in CD. It would, however, appear that cell depletion via CDC and/or ADCC is not a requisite for efficacy in this disease, as demonstrated by the positive clinical data from the PRECiSE studies.11, 12
Resistance of cells, such as T cells, to apoptosis has been proposed to play an important role in inflammatory bowel disease,18 and TNFα is a known survival factor for certain cell types. A number of published studies have examined the ability of the anti-TNFα agents to mediate apoptosis, in particular infliximab and etanercept. The data suggest that infliximab is capable of inducing apoptosis in human T cell and monocytic cell lines,4, 6 activated peripheral blood T cells or lymphocytes from normal donors,5, 19 lamina propria T cells,5, 6 and monocytes from patients with CD.3 Adalimumab was also observed to induce apoptosis of PBMC.4 In contrast, etanercept was found to be less effective at inducing apoptosis in PBMC, activated peripheral blood lymphocytes from normal donors, and lamina propria T cells from patients with CD,4, 5 suggesting that induction of apoptosis could be a key mechanism of action for anti-TNFα agents in CD. The data generated in our experimental systems, however, suggest that induction of apoptosis via binding to mTNFα may not be required for clinical efficacy in CD. Consistent with published reports, the proportion of activated T cells and monocytes from normal donors showing binding of Annexin V increased following incubation with either infliximab or adalimumab. Exposure to etanercept did result in an increased proportion of both cell types binding Annexin V, but at a reduced level compared with infliximab and adalimumab. In contrast to the other agents, certolizumab pegol did not induce any detectable increase in the proportion of either cell type binding Annexin V. It is important to recognize that the ability of certolizumab pegol to induce apoptosis of lamina propria cells derived from patients with CD has not been addressed in these studies. Hence, the potential for a different result under these conditions cannot be excluded because there is some evidence that lamina propria cells are more susceptible to infliximab-induced apoptosis.20 Whether any of these cell-killing mechanisms occur in vivo is also unclear, although it has been shown21–23 that there is a prolonged reduction in the numbers of PBMC after infliximab treatment.
It is not clear why certolizumab pegol does not induce apoptosis of cells bearing mTNF, as do the other anti-TNF agents. It may be because certolizumab pegol binds to a different epitope from the other agents, which leads to a different signaling pattern inside the cell. An increase in apoptosis has been reported in tissue sections from diseased bowel 24 hours6 and 28 days20 after infliximab treatment, although the cause of the increase in apoptosis is difficult to determine. It is possible that in this situation the anti-inflammatory effect of infliximab leads indirectly to apoptosis of activated cells.
As neutrophils commonly occur at sites of inflammation and are able to produce TNFα upon stimulation,24 we also examined the ability of the anti-TNFα agents to bind to human PMNs. After demonstrating that all 4 agents bound to the surface of these cells, functional effects resulting from binding were examined. Although certolizumab pegol bound to human PMNs, no increase in cell death or release of MPO was observed (Fig. 6), possibly because of a difference in the way in which it signals through membrane TNF. In contrast, the other 3 anti-TNFα agents all induced both cell death and release of MPO.
The implications of these findings are currently unclear. Previous studies investigating the effect of infliximab,25, 26 adalimumab,27 or etanercept26, 28 on neutrophil functionality did not report any significant functional alterations. However, these studies were conducted on the circulating neutrophil pool, and therefore did not assess potential decreases in the neutrophil population after dosing. Some evidence for decreases in circulating neutrophil numbers has been reported after treatment with infliximab.20, 29
There is evidence that mTNFα can mediate reverse signaling back into the cell30, 31 and this is a potential mechanism by which infliximab, adalimumab, and etanercept increase the proportion of apoptotic cells. A potential role for reverse signaling has been proposed in the subsequent downregulation of cytokine production in response to LPS stimulation8, 19, 32 by monocytes. Intriguingly, in these studies incubation with infliximab resulted in subsequent inhibition of LPS-induced cytokine production, whereas incubation with etanercept did not.8 Comparable data have been generated in the present studies, in which certolizumab pegol also effectively inhibited subsequent LPS-induced cytokine production. Although production of TNFα is inhibited, any potential effects of carry-over of the antibodies were addressed by measuring IL-1β. The structural differences between certolizumab pegol and the other anti-TNFα agents may provide important insights into the mechanisms underlying these observations. Bacteria and bacterial products are increasingly thought to play a role in inappropriate activation of the immune system in CD.33 This provides a potential link between these in vitro findings and mechanisms that may underlie the disease process, particularly as etanercept neither effectively inhibits this process, nor has been shown to be efficacious in CD. LPS stimulation of cells of the innate immune system is a very powerful signal suggesting that the inhibition mediated by some of the anti-TNFα agents may be a potent anti-inflammatory mechanism. Further ex vivo studies are ongoing using cells from the inflamed bowel of patients with CD in order to determine the relevance of this effect in clinical disease.
In summary, a series of comparative in vitro studies was conducted to explore the mechanism of action of certolizumab pegol in CD. The unique structure of this molecule, coupled with positive clinical data in this disease indication, have provided important new information to increase our understanding of the key requirements for an anti-TNFα agent in CD. While these studies are all based on in vitro systems, they nevertheless suggest that mechanisms such as induction of apoptosis, CDC, and ADCC may not be required for clinical efficacy of an anti-TNFα agent in CD. If these mechanisms are not required for efficacy, further studies are required to determine whether they may, in fact, be associated with undesirable effects. In contrast, inhibition of bacterially stimulated cytokine production from macrophages may be a necessary function for an anti-TNFα agent to produce efficacy in CD. A better understanding of the differences in mechanisms of action between the available anti-TNFα agents could enable the selection of only those functions required for clinical efficacy in the development of the next generation of anti-TNFα agents.