Preclinical Efficacy and Immunological Safety of FR104, An Antagonist Anti-CD28 Monovalent Fabʼantibody, in Humanized Mice Models

Abstract

Antagonist anti-CD28 antibodies prevent T-cell costimulation and functionally differentiate from CTLA4Ig since they cannot block CTLA-4, PDL-1 and CD80/86-mediated coinhibitory signals. They demonstrated efficacy in suppressing effector T cells while enhancing regulatory T cells function and immune tolerance after organ transplantation. So far, however, blocking anti-CD28 antibodies devoid of any agonist or superagonist activity and showing clinically acceptable pharmacokinetic profile have not been developed. FR104 is a novel monovalent humanized Fab' anti-CD28 antibody fragment that was pegylated to improve elimination half-life. We have investigated its mechanism of action, immunological safety and preclinical efficacy in humanized mouse models. FR104 dose-dependently prevented human T cell proliferation and IL-2 secretion in vitro. Following the guidelines edicted after the ‘Tegenero affair’ to exclude agonist activities of T-cell directed antibodies, we showed that FR104 failed to induce human T cell proliferation and cytokines secretion, even in the presence of anti-CD3 antibodies or when cross-linked with secondary antibodies, whereas superagonist and divalent anti-CD28 antibodies showed such activities. Furthermore, in humanized NOD/SCID mice adoptively transferred with human PBMC, superagonist and divalent anti-CD28 Abs elicited rapid cytokines secretion and human T-cell activation, whereas FR104 stayed antagonist. These humanized mice eventually developed a florid graft-versus-host disease due to the proliferation of xenogeneic human T cells, that was completely prevented by short-term administration of FR104. This effect was dependent on the availability of CTLA-4 since co-administration of an anti-CTLA-4 antibody abrogated the therapeutic activity of FR104. Interestingly, administration of high doses of Belatacept was ineffective to prevent graft-versus-host disease, whereas a less intensive regimen of administration was partially effective. In conclusion, we demonstrated in vitro and in vivo on human T cells that the anti-CD28 monovalent antibody FR104 is devoid of agonist activity and thus compatible with a clinical development that might lead to higher therapeutic indexes, by sparing CTLA-4, as compared to CD80/CD86 antagonists in autoimmune diseases and transplantation.