Abstract 5299: A novel anti-4-1BB antibody with no liver toxicity and its application in a bi-specific antibody
Authors: Chang Jen-Kuan , Xue Jie , Dong Chen , Ju An , Baral Toya , Yoo Julie , Wang Yanling , Muda Marco , Liu Qiang , Xu Wenfeng , Jiang Weidong , Feng Lixin 
Published: March 2024

Abstract
4-1BB (CD137, TNFRSF9) is a co-stimulatory receptor initially identified on T cells, belonging to the tumor necrosis factor receptor superfamily. Primarily expressed on activated CD8+ T cells, it plays a crucial role in immune activation. Moreover, the intracellular domain of 4-1BB is leveraged to enhance the proliferation and cytotoxicity of Chimeric Antigen Receptor-T (CAR-T) cells. However, the development of 4-1BB agonist antibodies has encountered challenges, with limited clinical efficacy (e.g., utomilumab) or dose-dependent liver toxicity (e.g., urelumab). Consequently, there is a significant demand for the development of next-generation 4-1BB agonist antibodies, leading to numerous ongoing preclinical R&D efforts and clinical trials. Here, we introduce a novel fully humanized anti-4-1BB antibody, HLX25, and discuss the development of HLX34, a bispecific antibody targeting both 4-1BB and Her2. HLX25 boasts a unique binding epitope distinct from first-generation 4-1BB antibodies and demonstrates multiple cross-species reactivities. Additionally, HLX25 incorporates an engineered Fc region to enhance intratumoral clustering while reducing toxicity. In vitro studies reveal that HLX25 exhibits activity similar to natural 4-1BB ligands. In contrast, utolimumab shows inadequate activity, and urelumab displays superior activity, suggesting a correlation between efficacy and safety. As reported previously, (As been profiled in our Lab,) HLX25 exhibits robust dose-dependent anti-tumor activity in the MC38/h4-1BB KI mouse model. Noteworthy is the superior safety profile of HLX25 in the h4-1BB KI model, where both HLX25 and utolimumab induce no significant AST and ALT upregulation, unlike urelumab, which demonstrates such upregulation in vivo. A comparative analysis of HLX25 with other second-generation anti-4-1BB antibodies in clinical trials in vivo reveals that HLX25 provides superior tumor inhibition without observed toxicity within the effective dose range. Furthermore, we've developed HLX34, a Her2x4-1BB bispecific antibody, derived from Trastuzumab and HLX25. Its format underwent optimization using a reporter assay, indicating superior in vitro activity. Validation through a cytokine release assay with primary PBMCs in vitro and confirmed in vivo activity in animal models, without observed toxicity, strongly supports the efficacy and safety of HLX34 in Her2-positive tumors. These results provide evidence that our novel anti-4-1BB antibody activates proper 4-1BB signaling through tumor-enriched FcgRIIB or tumor-associated antigen-mediated clustering, inhibiting tumor growth with good safety in both in vitro and in vivo settings. This underscores HLX34 as a promising alternative therapeutic strategy for next-generation cancer immunotherapy.
Citation Format: Jen-Kuan Chang, Jie Xue, Chen Dong, An Ju, Toya Baral, Julie Yoo, Yanling Wang, Marco Muda, Qiang Liu, Wenfeng Xu, Weidong Jiang, Lixin Feng. A novel anti-4-1BB antibody with no liver toxicity and its application in a bi-specific antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5299.