A Potent Single-Domain Antibody Targeting LAG-3 for Efficient Tumor Immunotherapy

Lymphocyte activation gene-3 (LAG-3) is a pivotal immune checkpoint receptor that exerts a negative regulatory effect on T-cell function. Although LAG-3-blocking antibodies have shown promising clinical potential, the inherent limitations of conventional monoclonal antibodies necessitate the development of novel antibody formats with enhanced biological and pharmacological properties. In this study, a panel of single-domain antibodies (sdAbs) targeting human LAG-3 was generated via phage display technology. Among these candidates, 2H-G7 was identified as a high-affinity sdAb that binds to LAG-3 with an equilibrium dissociation constant (KD) in the nanomolar range. Notably, 2H-G7 potently blocks the interactions of LAG-3 with both of its key ligands, fibrinogen-like protein 1 (FGL1) and major histocompatibility complex class II (MHC-II). Its capacity to restore impaired T-cell function was validated by quantifying interleukin-2 (IL-2) secretion and CD69 expression in stimulated primary human peripheral blood mononuclear cells (PBMCs). Epitope mapping studies localized the binding site of 2H-G7 to the D1D2 extracellular domains of LAG-3, distinct from relatlimab, a clinically approved LAG-3-blocking antibody serving as the benchmark. In a xenogeneic mouse model of non-small-cell lung cancer (NSCLC), 2H-G7-Fc exhibited superior tumor growth inhibition efficacy compared with relatlimab. These findings demonstrate that 2H-G7 is a promising lead candidate for the development of next-generation LAG-3-targeted tumor immunotherapies.