INTRODUCTION:CD40 ligand (CD40L) blockade has demonstrated efficacy in experimental autoimmune models. However, clinical trials of hu5c8, an anti-human CD40L IgG1 antibody, in systemic lupus erythematosus (SLE) were halted due to an increased incidence of thrombotic events. This study evaluated CDP7657, a high affinity PEGylated monovalent Fab' anti-CD40L antibody fragment, to assess whether an Fc-deficient molecule retains efficacy while avoiding the increased risk of thrombotic events observed with hu5c8.METHODS:The potency and cross-reactivity of CDP7657 was assessed in in vitro assays employing human and non-human primate leukocytes, and the capacity of different antibody formats to activate platelets in vitro was assessed using aggregometry and dense granule release assays. Given the important role CD40L plays in regulating humoral immunity, in vivo efficacy was assessed by investigating the capacity of Cynomolgus monkeys to generate immune responses to the tetanus toxoid antigen while the potential to induce thrombotic events in vivo was evaluated after repeat dosing of antibodies to Rhesus monkeys. A PEGylated anti-mouse CD40L was generated to assess efficacy in the New Zealand Black/White (NZB/W) mouse model of SLE.RESULTS:CDP7657 dose-dependently inhibited antigen-specific immune responses to tetanus toxoid in Cynomolgus monkeys, and in contrast to hu5c8, there was no evidence of pulmonary thrombovasculopathy in Rhesus monkeys. Aglycosyl hu5c8, which lacks Fc receptor binding function, also failed to induce thrombotic events in Rhesus monkeys. In vitro experiments confirmed that antibody constructs lacking an Fc, including CDP7657, did not induce human or monkey platelet activation. A PEGylated monovalent Fab' anti-mouse CD40L antibody also inhibited disease activity in the NZB/W mouse model of SLE after administration using a therapeutic dosing regimen where mice received antibodies only after they had displayed severe proteinuria.CONCLUSIONS:These findings demonstrate for the first time that anti-CD40L antibodies lacking a functional Fc region do not induce thrombotic events in Rhesus monkeys and fail to activate platelets in vitro but, nevertheless retain pharmacological activity and support the investigation of CDP7657 as a potential therapy for systemic lupus erythematosus and other autoimmune diseases.

Background: Inhibitors of tumor necrosis factor a (TNF alpha) have demonstrated significant efficacy in chronic inflammatory diseases, including Crohn's disease (M). To further elucidate the mechanisms of action of these agents, we compared the anti-TNF alpha agents certolizumab pegol, infliximab, adalimumab, and etanercept in several in vitro systems.Methods: The ability of each anti-TNF alpha agent to neutralize soluble and membrane-bound TNFa; 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 beta production by human monocytes was measured in vitro.Results: All 4 agents neutralized soluble TNFa and bound to and neutralized membrane TNF alpha. 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 beta production was inhibited by certolizurnab pegol, infliximab, and adalimumab, but only partially inhibited by etanercept.Conclusions: In contrast to the other anti-TNFa agents tested, certolizurnab 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-TNFa agents in CD. As certolizurnab pegol, infliximab, and adalimumab, but not etanercept, almost completely inhibited LPS-induced IL-1 beta release from monocytes, inhibition of cytokine production may be important for efficacy of anti-TNF alpha agents in CD.

Purpose: Induction of apoptosis in lymphocytes and monocytes by signaling through membrane tumor necrosis factor (TNF) is a proposed mechanism of action for anti-TNFs in Crohn's disease (CD). However, certolizumab pegol, an Fc-free PEGylated Fab’ anti-TNF, is efficacious in CD but does not mediate an apparent increase in apoptosis in vitro. Although etanercept was initially reported not to cause apoptosis (and this was cited as the reason for its lack of efficacy in CD) three groups have now shown that etanercept does mediate apoptosis in a similar manner to infliximab and adalimumab. The precise mechanism of the apoptotic effect of some anti-TNFs and its importance to efficacy are unclear. This study investigated the apparent pro-apoptotic effects of adalimumab, etanercept, and infliximab in activated monocytes and lymphocytes. Methods: Positive selection with MACS beads was used to separate monocytes from human peripheral blood mononuclear cells. The negatively selected population was used as the lymphocyte preparation. Monocytes were activated with GM-CSF and interleukin-4 for 3 days; lymphocytes were activated with CD3/CD28 for 2 days. Anti-TNFs or appropriate controls were then added (final concentration: 10 μg/mL) for a further 24 hours. The ViaCount assay system (Guava Technologies) was used to ascertain absolute cell counts and cell viability. Results: The absolute number of apoptotic cells did not increase above the levels seen in the respective control samples after treatment of monocytes or lymphocytes with any of the anti-TNFs. However, adalimumab, etanercept, and infliximab reduced the number of viable monocytes (36.3%, 35.6%, and 38.3%, respectively) and lymphocytes (31.7%, 32.7%, and 32.5%, respectively) relative to controls. In contrast, certolizumab pegol did not cause a significant reduction in the number of viable cells compared with control treatment. Conclusion: The anti-TNFs adalimumab, etanercept, and infliximab do not appear to increase apoptosis in isolated monocytes and lymphocytes; instead, they reduce the proliferation of these cells. However, since certolizumab pegol treatment does not reduce cell proliferation, anti-proliferative activity against monocytes and lymphocytes does not seem to be required for efficacy in CD. The mechanism of this effect is unclear and requires further investigation. This research was funded by UCB.

Advances in immunotherapy through the development of chimeric, humanized and fully human monoclonal antibodies have improved treatment and reduced immunogenicity. These strategies produced biological anti-tumor necrosis factor-α therapies for inflammatory conditions, including Crohn´s disease and rheumatoid arthritis. Antibody fragments, including antigen-binding fragments, are a further advance in antibody-based therapy. Certolizumab pegol is a PEGylated humanized antigen-binding fragment of an anti-tumor necrosis factor-α monoclonal antibody. PEGylation extends the half-life and is compatible with subcutaneous administration, while humanization may reduce immunogenicity. Large-scale antigen binding fragment production is possible via microbial fermentation. This review details how the monoclonal antibody was generated, selected and humanized from the original parent murine antibody, and summarizes the pharmacological characteristics, efficacy and safety data that distinguish it from the full monoclonal ...

Purpose: Proinflammatory cytokines, such as interleukin-1ββ (IL-1ββ) are over-expressed by monocytes/macrophages in patients with Crohn's disease (CD). It has been suggested that anti-tumor necrosis factor αα (TNFαα) agents mediate an inhibitory effect on lipopolysaccharide (LPS)-induced cytokine production via membrane TNFαα signalling. This study compared LPS-stimulated production of IL-1ββ in the presence of certolizumab pegol (an antibody Fab' fragment conjugated with polyethylene glycol) and other anti-TNF agents. Methods: Monocytes were positively selected using CD14+ magnetic microbead-associated cell sorting (MACS) from peripheral blood mononuclear cells of normal human donors. Purified monocytes were pre-incubated for 1 hr with certolizumab pegol, etanercept, adalimumab, infliximab (100 μg/mL to 100 pg/mL) or a relevant control. After extensive washing, the monocytes were incubated — with or without LPS (100 ng/mL) — for 4 hours at 37°C. Supernatants were assessed for IL-1ββ (by enzyme-linked immunosorbent assay) and a range of chemokines, cytokines and other proteins (by Luminex). Results: LPS-stimulated production of IL-1ββ by monocytes appeared to be completely inhibited in a dose-dependent manner by certolizumab pegol, infliximab and adalimumab. In contrast, etanercept was much less efficient at mediating this activity, causing only partial inhibition of cytokine production. Certolizumab pegol was approximately 100-fold more potent than infliximab and adalimumab at inhibiting the release of IL-1ββ by monocytes. The effects of infliximab and adalimumab were similar. The Luminex analysis of a panel of cytokines and chemokines showed a range of effects with inhibition of IL-10 and IL-12 being the most profound. Again etanercept was not as potent as the other anti-TNFs agents at inhibiting these cytokines. Conclusions: Effective inhibition of IL-1ββ production was seen with certolizumab pegol; inhibition was more potent than with adalimumab or infliximab. Even at high concentrations of etanercept, inhibition of IL-1ββ production was only partial. These are in vitro data, however, the comparative trends in inhibition of cytokine production stimulated by bacterial products appear to reflect the clinical efficacy of these anti-TNF agents in CD. The potent inhibition by certolizumab pegol of cytokine production by monocytes may represent an important mechanism of action in CD.

Purpose: Biologics that inhibit the effects of tumour necrosis factor α (TNFα) have shown clinical benefit in a range of inflammatory diseases. Infliximab is an intravenously administered, chimeric full IgG1 monoclonal anti-TNF antibody for Crohn's disease (CD). Use of infliximab may be limited by the induction of antibodies to infliximab (ATI), subsequent loss of efficacy and occurrence of infusion reactions.1 Certolizumab pegol is a subcutaneously administered PEGylated Fab' fragment of a humanised anti-TNF antibody currently in an advanced stage of development for the treatment of CD. Patients who develop antibodies following treatment with infliximab may have an opportunity to switch to treatment with certolizumab pegol if the antibody response to infliximab does not cross-react with certolizumab pegol. The aim of this study was to determine if the antibody response elicited in patients to infliximab cross-reacts with certolizumab pegol. Methods: Plasma samples were collected from 20 patients who had an antibody response to infliximab. Samples were assayed using ELISA plates coated with certolizumab pegol and then blocked with 0.1% bovine serum albumin. Plasma from patients with ATI was then incubated on the plates. Following a wash step, biotinylated certolizumab pegol was added followed by streptavidin horseradish peroxidase. Colour was developed using 3,3',5,5'-tetramethylbenzidine (TMB) substrate and the absorbance read at 450 nm with reference at 630 nm. Results: No cross-reactivity with certolizumab pegol was detected in any of the plasma samples (n = 20). Conclusions: In patients suffering from Crohn's disease, no cross reaction was detected between ATI and certolizumab pegol. It should therefore be possible for patients with CD who have an antibody response to one anti-TNF to use another anti-TNF agent. Subcutaneous certolizumab pegol represents a possible alternative treatment for patients who develop antibodies after intravenous treatment with infliximab.

Purpose: Certolizumab pegol, a PEGylated Fab' fragment of an anti-TNF monoclonal antibody, has no Fc region. Adalimumab and infliximab are whole immunoglobulin (Ig) G1s and possess an Fc region. An Fc region allows placental crossing via specific neonatal Fc receptors, FcRn.1 This is important in Crohn's disease as it affects women of child-bearing age. This study is the first to compare placental transfer of murinised IgG1 and PEGylated Fab' versions of hamster anti-murine TNF antibody (TN3) in pregnant rats and assess milk concentrations. Methods: Pregnant rats (Sprague-Dawley) received 100 mg/kg iv of either TN3 IgG1 (2 groups [grps] of 6) or TN3 PEGylated Fab' fragment (2 grps of 6). Grp 1 for each agent was dosed on Day (D) 6 and D13 of gestation; Grp 2 on D15. In Grp 1, dam blood was collected every 2–3 days of gestation and fetal blood on D20. In Grp 2, dam blood was collected every 2 days of gestation and every 3–4 days post partum. Pup blood was taken on D7 post partum and milk from dams on D8 post partum. TN3 IgG1 and PEGylated Fab' concentrations were determined by sandwich enzyme-linked immunosorbent assay. Results: IgG1 and PEGylated Fab' had plasma elimination profiles as expected in dam samples; the Fab' having a slightly shorter half-life. In the PEGylated Fab' Grp 1, 2/5 fetal samples had concentrations of the Fab' 0.3% of that in dam plasma. In IgG1 Grp 1, all fetal samples had a mean IgG1 concentration 15.6% of that in dams. In PEGylated Fab' Grp 2, no Fab' was detected in pup plasma D7 post partum; 4/5 milk samples contained agent at 4.9% of the dam plasma concentration. In IgG1 Grp 2, the IgG1 concentration in pup plasma D7 post partum was twice that of dams; milk concentrations were 24% of those in dam plasma. Conclusions: In the fetuses of 5 rats given anti-TNF PEGylated Fab' fragment whilst pregnant, PEGylated Fab' was found at very low concentrations in 2 fetuses but was not detectable in the others. This suggests that anti-TNF PEGylated Fab' does not undergo active FcRn-mediated placental transport in rats. Biologically relevant IgG1 concentrations were found in fetuses of rats given IgG1, showing that IgG1 had crossed the placenta. Milk concentrations were much lower for PEGylated Fab' than for IgG1 on D8 post partum. These results may be important for women of child-bearing age needing anti-TNF treatment.