Computational design of multi-objective optimized peptide disrupting Tim-3 galectin9 checkpoint

T cell exhaustion remains a major obstacle to effective cancer immunotherapy. The immune checkpoint TIM-3 is a key driver of this dysfunction. Although blocking the TIM-3/Galectin-9 interaction holds therapeutic promise, clinical translation has been limited by tumor immune evasion mechanisms. Here, we developed a multi-constraint peptide generation algorithm that integrates electrostatic, hydrophobic, solvent accessibility, hydrogen bonding, volume, and polarity matching as key affinity determinants. Candidates achieving high composite scores are then subjected to molecular docking to evaluate binding poses and binding energies against the TIM-3 receptor pocket. Top-ranked candidates from docking subsequently undergo molecular dynamics simulations to assess binding stability and conformational fidelity. Ten candidate peptides were synthesized via solid-phase methods. Among them, the lead peptide AT3P19 effectively disrupted the TIM-3/Galectin-9 axis and restored CD8⁺ T cell function, as evidenced by increased production of IFN‑γ, IL‑2, and TNF‑α along with reduced apoptosis. To democratize this approach, we established an open web platform (MCSE, https://slb.hospitalstar.com:7008 ) that implements the full algorithm, enabling users to perform de novo peptide design and evaluation for immune checkpoint targets. This work establishes a physicochemical framework for rational design of immune checkpoint modulators, paving the way for next-generation peptide-based immunotherapies.