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.

1 The L6 myocyte cell line expresses high affinity receptors for calcitonin gene-related peptide (CGRP) which are coupled to activation of adenylyl cyclase. The biochemical pharmacology of these receptors has been examined by radioligand binding or adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation. 5 The pharmacological profile of the L6 CGRP receptor suggests that it most closely resembles sites on skeletal muscle, cardiac myocytes and hepatocytes. The L6 cell line should be a stable homogeneous model system in which to study CGRP mechanisms and pharmacology.

IntroductionCD40 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.MethodsThe 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.ResultsCDP7657 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.ConclusionsThese 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0757-4) contains supplementary material, which is available to authorized users.

Defects in the gene encoding Bruton's tyrosine kinase (Btk), normally expressed in B cells, cause X-linked agammaglobulinemia (XLA). The phenotype of XLA is characterized by a lack of circulating B cells and immunoglobulin. It has been suggested that B cell maturation from the pre-B cell stage to more mature stages is dependent on the appropriate expression of this gene. The Btk mRNA is expressed in B cells and myeloid cells, but protein expression in relation to B cell maturation has not been determined. Moreover, expression of the Btk protein has so far only been investigated in human Epstein-Barr virus-transformed B cell lines, and in murine splenocytes and B cell lines. We have developed an antiserum which recognizes the human Btk protein and shown that normal human tonsillar B cells, peripheral blood monocytes and myeloid cells express the protein, whereas tonsil-derived T cells do not. We also show that the protein is present in early and mature human B cell lines, but is absent in terminally differentiated plasma cell lines. Furthermore, expression is reduced or absent in three B lineage cell lines derived from two patients with defined genetic mutations in Btk and suffering from XLA.

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