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Antibodies targeting the CD40-CD40L pathway have great potential for treating autoimmune diseases like rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), and inflammatory bowel diseases (IBD). However, in addition to the known difficulty in generating a purely antagonistic CD40 antibody, the presence of CD40 and CD40L on platelets creates additional unique challenges for the safety, target coverage, and clearance of antibodies targeting this pathway. Previously described therapeutic antibodies targeting this pathway have various shortcomings, and the full therapeutic potential of this axis has yet to be realized. Herein, we describe the generation and characterization of BI 655064, a novel, purely antagonistic anti-CD40 antibody that potently neutralizes CD40-CD40L-dependent B-cell stimulation without evidence of impacting platelet functions. This uniquely optimized antibody targeting a highly challenging pathway was obtained by applying stringent functional and biophysical criteria during the lead selection process. BI 655064 has favorable target-mediated drug disposition (TMDD)-saturation pharmacokinetics, consistent with that of a high-quality therapeutic monoclonal antibody.

Mechanical injury to the skin results in activation of the complement component C3 and release of the anaphylatoxin C3a. C3a binds to a seven-transmembrane G protein-coupled receptor, C3aR. We used C3aR(-/-) mice to examine the role of C3a in a mouse model of allergic inflammation induced by epicutaneous sensitization with OVA. C3aR(-/-) mice exhibited an exaggerated Th2 response to epicutaneous but not to intraperitoneal sensitization with OVA, as evidenced by significantly elevated levels of serum OVA-specific IgG1 and significantly increased secretion of the Th2 cytokines IL-4, IL-5, and IL-10 by antigen-stimulated splenocytes. Presentation of OVA peptide by C3aR(-/-) APCs caused significantly more IL-4 and IL-5 secretion by T cells from OVA-T cell receptor (OVA-TCR) transgenic mice compared with presentation by WT APCs. C3a inhibited the ability of splenocytes, but not of highly purified T cells, to secrete Th2 cytokines in response to TCR ligation. This inhibition was mediated by IL-12 secreted by APCs in response to C3a. These results suggest that C3a-C3aR interactions inhibit the ability of APCs to drive Th2 cell differentiation in response to epicutaneously introduced antigen and may have important implications for allergic skin diseases.

Targeting CD40-CD40L interactions has been an interesting drug concept for the treatment of autoimmune diseases given this pathway’s role in the development of both humoral and cell mediated immune responses. While preclinical blockade of CD40L is well documented in various autoimmune animal models of disease, limited data exists for CD40 blockade due to lack of a truly antagonistic anti-mouse CD40 tool antibody (Ab). Here we describe the in vitro and in vivo characterization of a fully antagonistic anti-mouse CD40 monoclonal Ab (BI CD40-1); a chimeric rat Fv anti-mouse CD40 mAb engineered with a mouse IgG2a Fc containing mutations to abrogate Fcγ receptor binding. BI CD40-1 blocks molecular CD40-CD40L interactions (IC50 = 0.25 nM) and exhibits potent binding to CD40 expressed on mouse B cells (EC50 = 0.42 nM ± 0.08). In vitro profiling of BI CD40-1 using a mouse splenocyte proliferation assay confirmed potent antagonistic activity (IC50 = 0.27 nM ± 0.09) as well as absence of any agonistic properties (stimulation index < 2 @ 67 nM). Administration of BI CD40-1 to mice prior to ovalbumin (OVA) immunization resulted in dose-dependent blockade of OVA-specific IgG responses (100, 100, 74, 0% inhibition at 10, 3, 1, and 0.3 MPK; day 13) correlating with similar dose dependent receptor occupancy and inhibition of B cell activation as measured by ex vivo CD54 upregulation. Prophylactic dosing in cGVHD model of scleroderma showed dose dependent protection in disease development as seen by decreased dermal thickness, myofibroblast counts and collagen deposition. BI CD40-1 represents a novel fully antagonistic anti-mouse CD40 mAb, an important tool for mechanistic understanding of the therapeutic value of targeting of CD40 in inflammatory diseases.

Free intracellular magnesium (Mg2+) plays a critical role in diverse immune cell functions. Human individuals with a genetic deficiency in the magnesium transporter MagT1 present with XMEN disease, a X-linked immunodeficiency characterized by uncontrolled Epstein-Barr virus infection and neoplasia, uncovering a critical role for Mg2+ in the regulation of NK and CD8T cell function. To interrogate the role of MagT1 in the immune response, we generated conditional knockout mice utilizing a tamoxifen inducible (Cre-ERT2) system to genetically delete Magt1. Here, we characterized the requirement for MagT1 in immune cell function following in vivo genetic deletion. MagT1 is not required for lymphocyte homeostasis following tamoxifen deletion, as T and B cell populations were unperturbed in the spleen, lymph node and thymus following short and long-term tamoxifan treatment. Magt1-deficient CD4+ T cells were defective in response to antigenic challenge following Ova immunization in vivo, as the numbers of BRDU+ CD4+ T cells were reduced. This observation was not due to a defect in proliferation, as the percentage of BRDU+ CD4+ T cells was similar between wild-type and Magt1-deficient mice. In support of this finding, Magt1-deficient CD4+ T proliferation is normal in multiple in vitro proliferation assays. While this observation is inconsistent with human findings from X-MEN patients, differences in human and mouse physiology might contribute to this discrepancy. Future interrogation of MagT1 function in NK and CD8+ T cells could provide mechanistic insight into the manifestations observed in X-MEN disease.