RESULTS Selective CD28 blockade results in superior graft survival as compared with CTLA-4 Ig, where both CD28 co-stimulatory and CTLA-4 co-inhibitory signals are blocked

To test the hypothesis that selective CD28 blockade with preserved CTLA-4 co-inhibitory signals may result in enhanced prolongation of graft survival after transplantation, we made use of a novel recombinant domain antibody specific for CD28, which contains only the antigen-binding V variable domain and lacks an Fc domain. To test the efficacy of this anti-CD28 dAb in inhibiting donor-reactive T cell responses in a fully MHC disparate model of endogenous polyclonal alloreactivity, B6 recipients of BALB/c skin grafts were treated with CTLA-4 Ig or CD28 dAb in the presence of anti-CD154 mAb (Fig. 1 A). As previously reported (Ford et al., 2007), the median survival time (MST) of animals treated with CTLA-4 Ig and anti-CD154 was 32 d, significantly longer than that of untreated animals (MST 14 d). In contrast, 100% of the animals treated with the CD28 dAb in the presence of anti-CD154 exhibited graft survival of> 50 d. To compare the effects of selective CD28 blockade in the absence of the additional immune modulation provided by the anti-CD154, we assessed skin graft rejection in a published model of minor antigen disparity (Fig. 1 B; Ehst et al., 2003). B6 recipients were grafted with skin from OVA-expressing transgenic donors, which results in rejection in control V dAb-treated animals with an MST of 19 d (Fig. 1 C; Ford et al., 2007). Rejection in CTLA-4 Ig-treated recipients was prolonged to 34 d. In contrast, treatment of graft recipients with the anti-CD28 dAb resulted in better long-term graft survival (MST> 100 d; Fig. 1 C). To confirm the specificity of this reagent for CD28, B6 splenocytes were incubated with increasing doses of an unlabeled control V dAb or an unlabeled anti-CD28 dAb, and then stained with a FITC-conjugated anti-CD28 mAb (clone E18) that competes with anti-CD28 dAb for binding to CD28. Results demonstrated a titratable reduction in fluorescence with increasing concentrations