Abstract 3420: Coordinated stimulation of APC and T cell functions by first-in-class, tri-specific AN8025 for next-generation cancer immunotherapy

Abstract Background: Immune checkpoint inhibitors (ICI), the cornerstone of current immunotherapies, primarily target T cells to reinvigorate the immune system against tumors. Recently, combinatory approaches, particularly in the form of bi-specific molecules such as Akeso’s PD-1/VEGF and Innovent’s PD-1/IL2, have garnered significant interest for their potential to deliver superior clinical efficacy beyond the ICI era. Building on this paradigm, AN8025 emerges as a tri-specific antibody fusion protein derived from a clinically validated and approved anti-PD-L1 antibody, fused with functionally optimized extracellular domain of CD86 and LAG3 on its C- and N-termini, respectively. Here we report that AN8025 leverages the LAG3 component to stimulate the differentiation and maturation of antigen-presenting cells (APCs), while the CD86 and anti-PD-L1 components synergistically amplify tumor-specific T cell responses. Together, these actions orchestrate a multifaceted enhancement of immune function. Method: The in-vitro binding activities of AN8025 to corresponding targets and targets expressed on cell lines or primary cells were assessd by BLI, ELISA, or flow cytometry. Its in-vitro functional activities were measured using both reporter and primary immune cells. In-vivo efficacy was evaluated in MC38-hPD-L1 syngeneic model and hPBMC-engrafted NOG mouse bearing the MDA-MB-231 model. The developability of AN8025 was assessed under various stress conditions, and the preliminary pharmacokinetic (PK) profile was characterized in SD rats and Cynomolgus monkeys. Results: AN8025 exhibited low single-digit nanomolar binding affinity to PD-L1 and CTLA4, while demonstrating over ten nanomolar binding to CD28. It effectively blocked the PD-1/PD-L1 interaction and the interaction between CTLA4 and CD80 proteins. Functionally, AN8025 demonstrated superior activation of T cells compared to the PD-L1 antibody alone and the combination of individual components of AN8025 (PD-L1 antibody, CD86-Fc, and LAG3-Fc). Significantly, the heightened T cell activation, primarily driven by the interaction of CD86 and CD28, is strickly dependent on the presence of PD-L1. In addition, AN8025 promoted the differentiation and maturation of antigen-presenting cells (APCs), as indicated by morphological changes, increased secretion of cytokines and chemokines, and induced expression of APC activation/maturation markers such as CD80/CD83. In-vivo, AN8025 alone showed superior efficacy compared to either ICI or the combination of individual components of AN8025. Furthermore, AN8025 exhibited favorable in-vitro stability and in-vivo pharmacokinetic profiles, with a long serum half-life in rats and in monkeys. Conclusion: AN8025 demonstrates superior in-vitro and in-vivo activities compared to ICIs, supporting its further preclinical and clinical development. Citation Format: Youping Wang, Wenjing Yang, Wanwan Chen, Kun Zhu, Zhihong Liu, Guolong Xu, Nanhai He. Coordinated stimulation of APC and T cell functions by first-in-class, tri-specific AN8025 for next-generation cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3420.