LAGi-DEL的发现与验证：一种纳摩尔级LAG-3抑制剂，可在肿瘤-免疫共培养模型中逆转免疫抑制

　　本研究发现并验证了LAGi-DEL——首个纳摩尔级别的小分子LAG-3抑制剂，通过DNA编码化合物库（DEL）筛选获得，其直接结合LAG-3（Kd达97 nM）并有效阻断LAG-3/MHC-II互作（EC50为138 nM）。研究证实LAGi-DEL可恢复T细胞活性、提升IFN-γ分泌，并在急性髓系白血病和肺癌共培养模型中促进免疫介导的肿瘤杀伤，效果媲美FDA批准的抗体药物relatlimab。其优异的类药性质（高溶解性、良好稳定性、低毒性等）为开发口服LAG-3抑制剂提供了全新蓝图。

　　

Highlight

LAGi-DEL represents the first nanomolar small molecule LAG-3 inhibitor identified through screening a 4.2-billion compound DNA-encoded library (DEL). It binds directly to LAG-3 with a K_d of 97 nM (SPR) and disrupts the LAG-3/MHC-II interaction with an EC₅₀ of 138 nM—the highest potency reported for a small molecule against LAG-3. Molecular docking and dynamics simulations reveal that LAGi-DEL induces stabilizing conformational changes within the LAG-3 binding pocket. Functionally, LAGi-DEL restores T cell activation, elevates IFN-γ secretion, and promotes immune-mediated killing in acute myeloid leukemia and lung cancer co-culture models, matching the efficacy of the FDA-approved antibody relatlimab. Pharmacokinetic profiling shows favorable drug-like properties, including high solubility, acceptable permeability, robust metabolic and plasma stability, low cytotoxicity toward normal cells, and negligible hERG and CYP liabilities.

Results and discussion

To identify novel LAG-3-targeted small molecules, we conducted an affinity-based selection campaign using the DELopen platform from WuXi AppTec, comprising approximately 4.2 billion DNA-encoded compounds (Figure 2A). The selection strategy involved immobilizing recombinant human LAG-3 protein onto Ni-NTA magnetic beads, which were then incubated with the DEL pool to allow potential binders to associate with the target. In parallel, control selections using beads lacking LAG-3 were carried out to account for non-specific binders. After multiple rounds of selection and amplification, enriched compounds were identified via high-throughput sequencing and cheminformatic analysis.

Conclusion

In this study, we report the discovery, functional validation, PK investigation and cellular characterization of LAGi-DEL, a nanomolar small-molecule LAG-3 inhibitor identified through DEL-screening a 4-billion DNA-encoded chemical library. LAGi-DEL demonstrated direct binding affinities toward LAG-3 (K_d) of 97 nM in SPR and disrupted the LAG-3/MHC-II interaction with an EC₅₀ of 138 nM. Furthermore, LAGi-DEL exhibited functional immune restoration in vitro through enhanced IFN-γ secretion and T-cell-mediated tumor killing in co-culture models of acute myeloid leukemia and non-small cell lung cancer. Its favorable pharmacokinetic profile, including high solubility, metabolic stability, and low off-target risks, supports its potential as an orally bioavailable LAG-3 inhibitor and a blueprint for next-generation immune checkpoint therapeutics.