HFB301001, an OX40-based immunotherapy, drives Treg clearance and CTL activation through optimized OX40 receptor clustering.

BACKGROUND: OX40, a key co-stimulatory receptor that amplifies T cell-mediated anti-tumor immunity, is a promising immunotherapeutic target. Despite most reported OX40 agonists in clinical trials having high affinity, the relationship between affinity and agonistic activity remains complex, necessitating further clarification of affinity's impact on OX40-based immunotherapy efficacy and its underlying mechanisms. METHODS: We generated the different affinity OX40 agonist antibodies were generated by phage display. Antibody-receptor interactions were modeled using AI-based prediction and validated by hydrogen-deuterium exchange. We assessed the receptor clustering, T cell activation, and regulatory T cell (Treg) depletion effect of OX40 antibodies with different affinities by confocal microscopy and reporter cell assays. We further evaluated the anti-tumor efficacy in multiple murine tumor models. The effects of HFB301001 treatment on tumor-infiltrating T cells, safety in cynomolgus monkeys, and immune activation in clinical samples were investigated using single-cell RNA sequencing (scRNA-seq), flow cytometry, ELISpot, and immunofluorescence. RESULTS: We identified the low-affinity OX40 agonist antibody HFB301001 and generated variants with different affinities via phage display. Compared with its high-affinity mutant, HFB301001 induced stronger receptor clustering, enhanced T cell activation, and mediated more potent natural killer-mediated antibody-dependent cell-mediated cytotoxicity for Treg depletion than its high-affinity mutant in vitro. Consistently, HFB301001 outperformed the high-affinity mutant by boosting intratumoral T cell infiltration/activation and Treg clearance in vivo. Additionally, HFB301001 exhibited favorable safety in cynomolgus monkeys and effectively activated tumor-infiltrating T cells in a clinically relevant tumor slice culture system. CONCLUSIONS: The reduced-affinity strategy represents a promising framework for the clinical development of OX40-targeted cancer immunotherapies. Currently, HFB301001 is concluding in a phase I clinical study involving patients with advanced solid tumors (NCT05229601).