Engineering of a novel anti-CD40L domain antibody for treatment of autoimmune diseases.

CD40-CD40L interactions play a critical role in regulating immune responses. Blockade of CD40L by Abs, such as the anti-CD40L Ab 5c8, demonstrated positive clinical effects in patients with autoimmune diseases; however, incidents of thromboembolism (TE) precluded further development of these molecules. In this study, we examined the role of the Fc domain interaction with FcγRs in modulating platelet activation and potential for TE. Our results show that the interaction of the 5c8 wild-type IgG1 Fc domain with FcγRs is responsible for platelet activation, as measured by induction of PAC-1 and CD62P. A version of 5c8 with a mutated IgG1 tail was identified that showed minimal FcγR binding and platelet activation while maintaining full binding to CD40L. To address whether Fc effector function is required for immunosuppression, a potent Ab fragment, termed a "domain Ab" (dAb), against murine CD40L was identified and fused to a murine IgG1 Fc domain containing a D265A mutation that lacks Fc effector function. In vitro, this dAb-Fc demonstrated comparable potency to the benchmark mAb MR-1 in inhibiting B cell and dendritic cell activation. Furthermore, the anti-CD40L dAb-Fc exhibited a notable efficacy comparable to MR-1 in various preclinical models, such as keyhole limpet hemocyanin-induced Ab responses, alloantigen-induced T cell proliferation, "heart-to-ear" transplantation, and NZB × NZW F1 spontaneous lupus. Thus, our data show that immunosuppression and TE can be uncoupled and that a CD40L dAb with an inert Fc tail is expected to be efficacious for treating autoimmune diseases, with reduced risk for TE.

A monovalent anti-human CD28 domain antibody antagonist: preclinical efficacy and safety.

Targeting the CD28-CD80/86 pathway with an anti-CD28 antagonist is a promising alternative to current therapies for autoimmunity. However, attempts at generating conventional anti-CD28 mAbs lacking stimulatory activity has been challenging. In this study, we describe anti-human CD28 receptor antagonist domain Abs (dAbs) that are specific for human CD28. These dAbs are potent inhibitors of T cell activation, with an EC50 of 35 ± 14 ng/ml for inhibition of proliferation. The EC50 of 53 ± 11 ng/ml in an ex vivo CD28 receptor occupancy assay corresponds with in vitro functional activity, suggesting a direct correlation. The anti-CD28 dAb is equipotent in the inhibition of CD80- and CD86-mediated T cell proliferation and does not interfere with CTLA-4-mediated downmodulation of CD86 expression on APCs. The anti-CD28 dAbs are monomeric and do not demonstrate any evidence of agonism or costimulatory activity. In cynomolgus monkeys, the anti-CD28 dAb demonstrated pharmacodynamic activity, as measured by the inhibition of a T cell-dependent Ab response, without evidence of T cell depletion or cytokine release. Furthermore, there was a strong correlation between systemic exposure, duration, and extent of CD28 receptor occupancy, and pharmacodynamic activity. Taken together, these data support clinical evaluation of this novel anti-CD28 dAb for the treatment of autoimmune diseases.

Drug-eluting stents: sirolimus and paclitaxel differentially affect cultured cells and injured arteries.

Sirolimus and paclitaxel eluted from stents inhibit cell proliferation and other cellular processes by dramatically different mechanisms. In this study, the effects of sirolimus and paclitaxel on cultured human coronary artery smooth muscle and endothelial cell function or cell cycle changes in balloon-injured arteries were directly compared. Both sirolimus and paclitaxel inhibited smooth muscle and endothelial cell proliferation. However, only paclitaxel inhibited smooth muscle and endothelial cell migration at low (nM) concentrations. Sirolimus arrested smooth muscle and endothelial cells in the G0/G1 phase of the cell cycle without inducing apoptosis while paclitaxel produced apoptosis in both cell types at low nanomolar concentrations. Although both agents blocked neointimal formation, sirolimus applied locally to injured rat carotid arteries increased the percentage of cycling vascular cells in G0/G1 without inducing apoptosis while paclitaxel increased the percentage of cycling cells in S and G2/M phases while inducing apoptosis. These results suggest that sirolimus reduces neointimal hyperplasia through a cytostatic mechanism while paclitaxel produces apoptotic cell death.