A PEGylated Fab' fragment against tumor necrosis factor for the treatment of Crohn disease: exploring a new mechanism of action.

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)-alpha is a multifunctional cytokine involved in the regulation of immune responses. Elevated levels of TNFalpha are found in a wide range of diseases, including the chronic inflammatory conditions rheumatoid arthritis, psoriasis, and Crohn disease (CD). Anti-TNFalpha antibodies have proved highly efficacious in the treatment of these conditions. In addition, they have proved invaluable for investigating the role of TNFalpha in disease etiology. Based on evidence showing that neutralizing antibodies to TNFalpha were effective in animal models of CD, anti-TNFalpha antibody treatments were assessed in clinical trials. Interestingly, the anti-TNFalpha antibody etanercept proved ineffective at achieving remission in active CD despite potently neutralizing soluble TNFalpha. This indicated that an additional mode of action is also involved in the efficacy of the anti-TNFalpha 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-TNFalpha 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-TNFalpha 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.

Mechanism of action of certolizumab pegol (CDP870): in vitro comparison with other anti-tumor necrosis factor alpha agents.

BACKGROUND: Inhibitors of tumor necrosis factor alpha (TNFalpha) 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-TNFalpha agents certolizumab pegol, infliximab, adalimumab, and etanercept in several in vitro systems. METHODS: The ability of each anti-TNFalpha agent to neutralize soluble and membrane-bound TNFalpha; 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)-1beta production by human monocytes was measured in vitro. RESULTS: All 4 agents neutralized soluble TNFalpha and bound to and neutralized membrane TNFalpha. 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-1beta production was inhibited by certolizumab pegol, infliximab, and adalimumab, but only partially inhibited by etanercept. CONCLUSIONS: In contrast to the other anti-TNFalpha 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-TNFalpha agents in CD. As certolizumab pegol, infliximab, and adalimumab, but not etanercept, almost completely inhibited LPS-induced IL-1beta release from monocytes, inhibition of cytokine production may be important for efficacy of anti-TNFalpha agents in CD.

The use of surrogate antibodies to evaluate the developmental and reproductive toxicity potential of an anti-TNFalpha PEGylated Fab' monoclonal antibody.

Certolizumab pegol (CZP) is a PEGylated Fab' fragment of a humanized monoclonal immunoglobulin G (IgG)1 antibody that binds to human tumor necrosis factor alpha (TNFα) with high affinity. As for many monoclonal antibodies (mAbs), nonclinical safety assessment of CZP has been constrained because of its limited species cross-reactivity and recognition of only nonhuman primate and human TNFα, which presents particular challenges for assessing reproductive and developmental safety. To comprehensively assess the potential liability of TNFα suppression on reproductive and developmental processes, a PEGylated Fab' anti-rat TNFα antibody surrogate (cTN3 PF) has been developed and evaluated for reproductive toxicity. Conventional rat fertility and early embryonic development, embryo-fetal toxicity and pre- and postnatal development studies have been shown to be free of maternal, reproductive, or development toxicity effects, following sustained TNFα inhibition with cTN3 PF. Importantly, these studies have also shown that in marked contrast to a whole IgG anti-TNFα antibody, the PEGylated Fab' antibody cTN3 PF homologous to certolizumab pegol demonstrated negligible fetal exposure following maternal administration during the period of organogenesis. In addition to minimal placental transmission, transfer to milk was lower and fetal absorption negligible compared with the whole IgG antibody cTN3 γ1, resulting in little or no detectable antibody in the plasma of lactating pups.