Preclinical characterization of IBD0333, a novel CD24×4-1BB bispecific antibody with tumor-dependent agonistic activity for cancer immunotherapy

Background The immunosuppressive nature of tumor microenvironment (TME) limits the efficacy of current immunotherapies. Bispecific antibodies (bsAbs) offer a promising strategy to overcome this challenge by enabling localized immune activation. Specifically, simultaneously targeting the "don't eat me" signal CD24 on tumor cells and the co-stimulatory receptor 4-1BB on immune cells may provide potent anti-tumor effects while mitigating the systemic toxicity typically associated with conventional 4-1BB agonists. Here, we present the preclinical evaluation of IBD0333, a novel bsAb targeting CD24 and 4-1BB. Methods The binding affinity of IBD0333 to CD24 and 4-1BB was evaluated, and its pharmacodynamic effects were assessed through multiple in vitro assays. Anti-tumor efficacy of IBD0333 was evaluated using C57BL/6J-h4-1BB humanized mouse models in vivo, while pharmacokinetic and safety profiles were further investigated in cynomolgus monkeys. Results IBD0333 exhibited high-binding affinity to both human and cynomolgus monkey CD24 and 4-1BB proteins. Its agonistic activity toward 4-1BB was strictly dependent on CD24, as evidenced by potent NF-κB activation and cytokine induction only in the presence of CD24-positive tumor cells. IBD0333 also demonstrated appropriate FcγR binding and no-detectable ADCC/CDC activity. In a syngeneic tumor model, complete tumor regression was achieved at doses of 1 mg/kg and 3 mg/kg. Furthermore, IBD0333 was well tolerated in cynomolgus monkeys with no observed immunotoxicity or hepatotoxicity, and the no-observed-adverse-effect level (NOAEL) following repeated administration was established as 120 mg/kg. Conclusion IBD0333 is a tumor-conditionally activated anti-CD24×4-1BB bsAb that demonstrates potent antitumor efficacy along with a favorable safety and pharmacokinetic profile. These findings support its further clinical development.