Fc-Optimized Agonistic Anti-Human CD40 mAb Demonstrates Strong Antitumor Activity in hCD40/hFcγRIIB Dual Humanized Mice

Abstract As a promising antitumor immunotherapy, the agonistic anti-CD40 antibody is limited by its clinical response and toxicity. The selective binding of anti-CD40 antibodies to FcγRIIb has been reported to improve the anti-tumor efficacy. Therefore, we developed the CD40 single humanized and CD40/FcγRIIB dual humanized mouse model, and characterized the expression and pathway activation of chimeric h/m CD40 and FcγRIIB by FACS. Moreover, to evaluate the efficacy and toxicity of anti-hCD40 in vivo, the hCD40 and hCD40/hFcγRIIB mice were engrafted with MC38 syngeneic tumors and treated with anti-human CD40 antibody (MIL97) with mouse or human Fc domain. The binding epitope of MIL97 has a strong potential to rely on cross-linking CD40 signaling activation, and the functional modification of Fc enhances the binding to FcγRllB, resulting in stronger cross-linking and agonistic effects on CD40 signaling. Notably, in comparison with MIL97-mFc, the MIL97 with human Fc domain failed to inhibit the tumor growth in hCD40 mice, but led to ~50% tumor growth inhibition in MC38 tumors in hCD40/hFcγRIIB dual knockin mice. Meanwhile, we observed the toxicity indicated by the body weight change and liver function damage of the MIL97-hFc in hCD40/hFcγRIIB mice, mirror clinical toxicities. Collectively, the hCD40/hFcγRIIB mice provide a powerful preclinical model to predict the activity and safety of anti-hCD40 antibodies in vivo, especially of human-specific Fc-engineered antibodies.