Design and Biophysical Characterization of Second-Generation Cyclic Peptide LAG-3 Inhibitors for Cancer Immunotherapy

Lymphocyte activation gene 3 (LAG-3) is an inhibitory immune checkpoint crucial for suppressing the immune response against cancer. Blocking LAG-3 interactions enables T cells to recover their cytotoxic capabilities and diminishes the immunosuppressive effects of regulatory T cells. A cyclic peptide (Cys-Val-Pro-Met-Thr-Tyr-Arg-Ala-Cys, disulfide bridge: 1-9) was recently reported as a LAG-3 inhibitor. Based on this peptide, we designed 19 derivatives by substituting tyrosine residue to maximize LAG-3 inhibition. Screening via TR-FRET assay identified 8 outperforming derivatives, with cyclic peptides 12 [Tyr6(L-3-CN-Phe)], 13 [Tyr6(L-4-NH2-Phe)], and 17 [Tyr6(L-3,5-DiF-Phe)] as top candidates. Cyclic peptide 12 exhibited the highest inhibition (IC50 = 4.45 ± 1.36 μM). MST analysis showed cyclic peptides 12 and 13 bound LAG-3 with KD values of 2.66 ± 2.06 μM and 1.81 ± 1.42 μM, respectively, surpassing the original peptide (9.94 ± 4.13 μM). Docking simulations indicated enhanced binding for cyclic peptide 12, with a docking score of -7.236 kcal/mol compared to -5.236 kcal/mol for the original peptide.