No abstract

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.

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.

No abstract

The binding specificity of the murine OKT3 has been transferred into a human antibody framework to reduce its immunogenicity. This "humanized" anti-CD3 mAb (gOKT3-5) was previously shown to retain, in vitro, all the properties of native OKT3, including T cell activation, which has been correlated, in vivo, with the severe side effects observed in transplant recipients after the first administration of the mAb. T cell activation is thought to be triggered by the cross-linking mediated by the antibodies between T cells and Fc receptor-bearing cells. In this study, we introduced a single amino acid mutation from a leucine to a glutamic acid at position 235 in the Fc receptor binding segment of the gOKT3-5 mAb to produce Glu-235 mAb. This mutation generated a 100-fold decrease in the affinity of the antibody for the Fc receptor on U937 cells, without affecting Ag binding. In parallel, we observed a marked reduction in the T cell activation triggered by the mAb (proliferation, cell surface expression of early activation markers including Leu 23 and IL-2R, and release of TNF-alpha, IFN-gamma, and granulocyte macrophage-CSF). In contrast, the mutated mAb retained suppressive properties similar to the gOKT3-5 mAb, as assessed by significant modulation of the T cell receptor complex and suppression of Ag-specific CTL activity. We conclude that this anti-CD3 mAb bearing a single amino acid mutation in its Fc portion retains important immunosuppressive properties, while exhibiting significantly less T cell activation than OKT3 in vitro. This drug might achieve potent immunosuppression while minimizing acute toxicity in vivo and thus be useful in transplantation as well as in autoimmune diseases.