Apigenin is a direct small-molecule inhibitor of LAG-3 that synergizes with PD-L1 blockade for cancer immunotherapy

<title>Abstract</title> <p> Background Immune checkpoint blockade targeting PD-1/PD-L1 and LAG-3 has demonstrated significant clinical benefit. The recent FDA approval of combined LAG-3 and PD-1 blockade (relatlimab plus nivolumab) shows the clinical value of dual checkpoint inhibition. Current strategies rely almost exclusively on monoclonal antibodies, and the development of small-molecule modulators of LAG-3 remains largely unexplored. Here, we investigate whether apigenin, a naturally occurring flavonoid with reported anticancer and immunomodulatory properties, can serve as a small-molecule alternative to anti-LAG-3 antibodies in combination immunotherapy. Methods Flavonoids were screened for disruption of the LAG-3/MHCII interaction using HTRF. Binding of apigenin was validated by molecular dynamics, microscale thermophoresis and chemoproteomic pull-down. Functional activity and combination effects with the anti-PD-L1 antibody durvalumab were assessed in Jurkat reporter assays and human PBMCs, with synergy quantified using Bliss independence modeling. A polymeric formulation was developed to improve solubility and reduce nonspecific cytotoxicity. Antitumor efficacy was evaluated in a syngeneic MC38 mice model expressing human PD-L1. Results Apigenin disrupted the LAG-3/MHCII interaction and directly bound LAG-3 in cells, without detectable activity against PD-1/PD-L1. Functionally, formulated apigenin restored T-cell activation and worked synergistically with durvalumab, yielding uniformly positive Bliss-independence scores (peaking at ΔBliss ≈ 0.20) within a non-toxic concentration range enabled by formulation. In primary human PBMCs, the combination increased CD69 and CD137 expression and raised IFN-γ production. Quantitative proteomics showed a coordinated reprogramming of T-cell programs, with up-regulation of immune-activation and leukocyte-migration pathways and down-regulation of extracellular-matrix and tissue-remodeling programs associated with immune exclusion. In vivo, combination therapy significantly improved tumor growth inhibition, increased the intratumoral CD8 ⁺ T-cell frequency, and raised granzyme B and perforin expression without detectable toxicity. Conclusions Apigenin is a direct small-molecule inhibitor of LAG-3 that disrupts the LAG-3/MHCII interface and synergizes with PD-L1 blockade to increase antitumor immunity. These findings show that an inhibitory immune receptor traditionally addressed only by biologics can be engaged by a small molecule, and support antibody–small-molecule combinations as a strategy for dual checkpoint immunotherapy. </p>