A humanized anti-b7h3×4-1BB bispecific antibody exerts potent antitumour effects through the activation of innate and adaptive immunity

Agonistic monoclonal antibodies targeting 4-1BB have shown much preclinical promise, but their clinical development has been limited by obvious toxicity or unremarkable efficacy. Here, we generated two humanized anti-B7H3 × 4-1BB bsAbs (HK056-001/002) by fusing an anti-4-1BB scFv to the C-terminus of an anti-B7H3 with an intact Fc fragment from human IgG1 or IgG4. The two bsAbs were able to stimulate the 4-1BB signaling pathway, which was strictly dependent on B7H3 expression. In particular, HK056-001 retained Fc function and induced an ADCC effect in tumor cells, whereas HK056-002 did not. Strikingly, HK056-001 showed superior antitumour activity to HK056-002 both in vitro and in vivo. HK056-001 enhanced antitumour immunity and induced lasting antigen-specific immune memory to prevent tumor regrowth upon rechallenge, even at a dose as low as 2 mg/kg. Furthermore, HK056-001 did not induce nonspecific production of proinflammatory cytokines and had no apparent ability to induce ADA production. In addition, HK056-001 has no significant liver toxicity in human 4-1BB-KI BALB/c mice bearing CT26-B7H3 tumors. The optimal anti-B7H3 × 4-1BB bsAb HK056-001 exhibited synergistic antitumour effects by inducing an ADCC effect (innate immunity) and activating the 4-1BB signaling pathway (adaptive immunity) upon cross-bridging with B7H3 with no obvious toxicity, which could potentially provide a better therapeutic window compared to what is seen with 4-1BB agonists.