Abstract HLX43 is an innovative PD-L1-targeted antibody-drug conjugate (ADC) designed for cancer treatment. It consists of HLX20, an engineered anti-PD-L1 humanized IgG1 antibody, conjugated via a protease-cleavable tripeptide linker to a potent camptothecin-derived topoisomerase I inhibitor (C24) at a drug-to-antibody ratio (DAR) of 8. Previously reported preclinical studies have demonstrated that HLX43 retains the binding affinity and internalization kinetics of the parental HLX20 antibody and exhibits favorable stability and potent antitumor activity. The mechanism of action (MoA) of HLX43 is multifaceted, integrating immune checkpoint blockade with payload-induced cytotoxicity. The preclinical studies reported here show that, as an anti-PD-L1 agent, HLX43 effectively blocks PD-1/PD-L1 interaction and reactivates T-cell activity, evidenced by increased IL-2 and IFNγ secretion in mixed lymphocyte reaction assays. The direct cytotoxicity of HLX43 arises from inhibition of DNA repairing and induction of cell apoptosis as evidenced by cleaved PARP and increased γH2AX. In addition, HLX43 induces immunogenic cell death (ICD) marked by increased cell-surface calreticulin, contributing to its immunomodulatory effects. In parallel, protease-mediated cleavage of the linker within the tumor microenvironment (TME)—by enzymes such as matrix metalloproteinases (MMPs) and cysteine proteases—enables payload release independent of the PD-L1 binding. The extracellularly released payload exerts a bystander effect by diffusing into neighbor tissues and killing adjacent PD-L1-negative cancer cells, a feature that underlies HLX43’s robust efficacy in patient-derived xenograft (PDX) models with heterogeneous PD-L1 expression. Beyond direct cytotoxicity, HLX43 also remodels the TME, with in vivo studies showing increased infiltration of cytotoxic CD8+ T cells and reduced regulatory T cells (Tregs). These CD8+ T cells display enhanced effector function, including elevated IFNγ and Granzyme B expression. In summary, HLX43’s convergent mechanisms—combining immune checkpoint inhibition, targeted cytotoxicity, and TME modulation—support its potential as both a monotherapy and in combination with other immuno-oncology agents, positioning HLX43 as a promising therapeutic candidate across diverse tumor types Citation Format: Lixin Feng, Jijun Yuan, Wan-Jen Yang, Chenqiang Jia, Ge Song, Qingyu Wang, Jun Zhu, . Preclinical studies reveal that protease-mediated payload release and immuno-oncology/immunomodulatory effects contribute to the antitumor activity of HLX43 abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5704.
Feng et al. (Fri,) studied this question.