A Peptide Inhibitor of Lymphocyte Activation Gene-3 Interaction with Fibrinogen-like Protein 1 Synergizes with Programmed Death-Ligand 1 Blockade to Restore T Cell Activity and Inhibit Tumor Growth.

The immune checkpoint lymphocyte activation gene-3 (LAG-3) interacts with major histocompatibility complex class II and fibrinogen-like protein 1 (FGL-1) to suppress T cell activity, contributing to resistance against immunotherapies such as programmed death-ligand 1 (PD-L1) blockade. This study aimed to identify LAG-3-binding peptides capable of disrupting these immunosuppressive interactions. Using a phage-displayed peptide library, we identified 2 candidate peptides, LAG3pep-1 and LAG3pep-2, that preferentially bound to LAG-3-expressing cells and recombinant LAG-3 protein. A LAG-3 D1 domain-blocking antibody competitively inhibited the peptide binding to LAG-3-expressing cells, confirming target specificity. Among the 2 peptides, LAG3pep-2 demonstrated higher binding affinity and greater stability toward LAG-3. These findings were further supported by structural modeling and all-atom simulation of the LAG-3 and peptide complexes and in silico mutational analysis. Functionally, LAG3pep-2 restored interleukin-2 secretion in T cells suppressed by tumor-derived FGL-1 more effectively than LAG3pep-1. Furthermore, LAG3pep-2 synergized with a PD-L1-blocking antibody to rescue T cell cytotoxicity and cytokine production in tumor cell co-culture assays. In vivo, systemic administration of LAG3pep-2 in combination with PD-L1 blockade significantly suppressed syngeneic tumor growth, enhanced anti-tumor immunity, and exhibited no observable systemic toxicity, outperforming either monotherapy. Collectively, these findings identify LAG3pep-2 as a peptide inhibitor of LAG-3 interaction with FGL-1 and a promising combinatorial agent to potentiate PD-L1-targeted cancer immunotherapies.