THU0407 Preclinical Characterization of a Highly Selective and Potent Antagonistic Anti-CD40 mAb

Abstract

BackgroundCostimulatory molecule pair CD40-CD40L plays a central role not only in immune cell interactions but also in immune-non immune cell activation. Targeting this pathway has generated great interest but early attempts to target CD40L failed mainly due to thrombotic complications observed in the clinic. In addition, developing a potent truly antagonistic CD40 antibody has proven to be challenging.ObjectivesHere we describe the preclinical characterization of BI 655064, an anti-human CD40 mAb that is being developed for the treatment of autoimmune disorders. BI 655064 is engineered as a human IgG1 molecule with a mutated Fc region to abrogate effector function.MethodsBinding of BI 655064 to CD40 expressed on primary B cells was measured by flow cytometry. The ability of BI 655064 to block CD40L-induced B cell proliferation in vitro was measured by tritium uptake, while blockade of endothelial and DC activation was measured by inhibition of cytokine release. A subcutaneous PK/PD study in the cynomolgus monkey was performed to establish correlations between BI 655064 exposure, target coverage using a receptor occupancy assay and pharmacodynamic effects using an ex vivo CD54-induction assay. In vivo blockade of B cell function was also tested in a human-peripheral blood lymphocyte (huPBL) induced SCID mouse model of graft-versus-host disease (GvHD) and in cynomolgus monkeys immunized with keyhole limpet hemocyanin (KLH).ResultsIn human whole blood, BI 655064 binds to CD40 on B cells (EC90 of 6.85 nM ±0.74). Blockade of CD40L-induced B cell proliferation was observed at an average IC50 of 0.4 nM. Inhibition of CD40L mediated cytokine release was observed in DCs (IC50=0.25 nM and 0.59 nM for TNFα and IL12/23p40, respectively) and to verify effects beyond immune cells, BI655064 was tested in CD40L stimulated endothelial cell cultures. Binding of BI 655064 to human platelets did not induce or augment platelet activation as determined by in vitro induction of CD62P. In the PK/PD study, cynomolgus monkeys dosed with greater than 1 mg/kg of BI 655064 exhibited complete blockade of ex vivo CD54 upregulation corresponding to full CD40 target coverage on B cells. In vivo, BI 655064 demonstrated clear effects on B cell function in the GvHD model where both human IgM and IgG responses were completely abrogated. As part of a repeat dose tolerability study, BI 655064 given to cynomolgus monkeys prior to immunization with KLH resulted in inhibition of KLH-specific IgM and IgG antibody responses. At doses of 5 mg/kg and above, germinal center size was decreased microscopically in Peyer's patches, lymph nodes, spleens and tonsils in treated monkeys. Evaluation of platelet function in cynomolgus monkeys dosed with BI 655064 did not reveal any effects on ex vivo platelet activation or aggregation.ConclusionsBI 655064 is a humanized antagonistic anti-CD40 mAb which is to be tested in human clinical trials for autoimmune disorders to establish safety and efficacy. BI 655064 demonstrated relevant pharmacologic in vitro and in vivo ac...

Safety, Pharmacokinetics, and Pharmacodynamics of Multiple Rising Doses of BI 655064, an Antagonistic Anti‐CD40 Antibody, in Healthy Subjects: A Potential Novel Treatment for Autoimmune Diseases

“…Previously, investigation into platelet aggregometry and binding studies with human platelets showed that blocking CD40 had no obvious impact on platelet function (unpublished data); in toxicology studies, it was demonstrated that BI 655064 bound to platelets in cynomolgus monkeys had no impact on platelet number or function . Taken together, these findings indicate that when bound to platelets, BI 655064 does not appear to alter platelet activation, aggregation, or function . These data, as well as data from other anti‐CD40 or anti‐CD40L antibodies lacking a functional Fc region, support the interpretation that the risk of thromboembolic events, as observed with earlier anti CD40L antibodies, can be avoided by eliminating the function of the Fc region of the antibodies.…”

“…In line with observations after administration of single IV and SC doses of BI 655064, no thromboembolic events were reported during multiple dosing, and there were no clinically relevant changes in platelet or coagulation parameters. Previously, investigation into platelet aggregometry and binding studies with human platelets showed that blocking CD40 had no obvious impact on platelet function (unpublished data); in toxicology studies, it was demonstrated that BI 655064 bound to platelets in cynomolgus monkeys had no impact on platelet number or function . Taken together, these findings indicate that when bound to platelets, BI 655064 does not appear to alter platelet activation, aggregation, or function .…”

“…BI 655064 demonstrated potent and comparable binding properties in both human (EC 90 = 6.85 ± 0.74 nM) and cynomolgus monkey B cells, and potent inhibition of CD40L‐induced peripheral blood mononuclear cell proliferation without agonism. When bound to platelets, BI 655064 does not appear to alter platelet activation, aggregation, or function . In preclinical assessments in cynomolgus monkeys, with multiple doses up to 50 mg/kg BI 655064 for 26 weeks, reversible decreases in B‐cell levels, reversible reduction of lymphoid organ germinal centers, and good general tolerability without thromboembolic events or relevant cytokine release were demonstrated ( 21 and unpublished data).…”

“…BI 655064 is a humanized antagonistic anti‐CD40 monoclonal antibody that selectively binds CD40 and blocks the CD40‐CD40L interaction; it was designed to have no agonistic activity and to prevent stimulating cytokine production . Two replacement mutations in the Fc region (Leu234Ala and Leu235Ala) were incorporated to prevent Fc‐mediated antibody‐dependent or complement‐mediated cellular cytotoxicity and platelet activation.…”

Effects of BI 655064, an antagonistic anti-CD40 antibody, on clinical and biomarker variables in patients with active rheumatoid arthritis: a randomised, double-blind, placebo-controlled, phase IIa study

“…BI 655064 is a humanised, antagonistic anti-CD40 monoclonal antibody that selectively binds CD40 and blocks the CD40–CD40L interaction;13 two mutations in the Fc region were introduced to prevent Fc-mediated antibody-dependent or complement-mediated cellular cytotoxicity and platelet activation 13. BI 655064 demonstrated potent and comparable binding properties in both human and cynomolgus monkey B-cells and did not cause platelet activation, aggregation or function 13–15…”