Bispecific anti-CD40LXCD28 antibody prevents mouse xenogeneic graft-versus-host disease

Background: Graft-versus-host disease (GVHD) is a systemic complication of transplantation, primarily arising from the activation of donor CD4+ T cells that recognize recipient cells as foreign.The secondary signals required for CD4+ T cell activation mainly rely on two co-stimulatory molecules, CD40L and CD28.Blocking either of these two signals can induce immunological tolerance to suppress the occurrence of GVHD.In this study, we developed an anti-CD40LXCD28 bispecific antibody (bsAb) and tested its efficacy in the prevention of GVHD.Methods: In an in vitro setting, we studied the affinity of anti-CD40LXCD28 bsAb to CD40L and CD28 and its efficacy in blocking CD40L-CD40 and CD28-CD80 interactions, as well as in inhibiting the activation of human T cells.Xenogeneic GVHD was induced in NSG mice (n=5/group) by injecting human peripheral blood mononuclear cells (PBMCs; 1.0107 cells/injection) bi-daily for three weeks.Anti-CD40LXCD28 bsAb or a combination of control anti-CD40L and anti-CD28 bsAbs (2 mg/kg) were injected following the same schedule.The body weight and GVHD scores of recipient mice were monitored for 8 weeks, and their changes were analyzed by two-way analysis of variance with Tukey's multiple-comparison test.Results: When left untreated, three of five PBMC-injected mice died during the monitoring period.In the mouse group treated with the combination of control bsAbs, significant body weight loss and elevated GVHD scores were observed compared to control PBMC-uninjected mice (P<0.05 and P<0.0001, respectively).In contrast, mice treated with the anti-CD40LXCD28 bsAb had no statistically significant changes in both body weight and GVHD scores compared to PBMC-uninjected controls, and demonstrated significantly reduced body weight loss and GVHD scores compared to the control bsAbs combination-treated mouse group (P=0.0001both).Conclusions: Anti-CD40LXCD28 bsAb effectively inhibited GVHD and was more potent compared to the combined treatment of anti-CD40L and CD28 antibodies.