Inter-crosslinking peptide augments 4-1BB receptor clustering for cancer immunotherapy

4–1BB as a co-stimulatory receptor represents a next-generation therapeutic target in immune-oncology, which has been targeted by agonist antibodies or embedded in CAR-T cell construction. Notably, 4–1BB activation requires cluster formation due to natural ligand association. However, precise and robust regulation of receptor clustering still encounters great challenges. Herein, we report a rationally designed self-assembled peptide ligand, composed of targeting and assembly modules, for in situ triggering ligand-receptor complex aggregation and augmenting clustering on T cells. The unique peptide ligand structure imparts ligand inter-crosslinking (LIC) effect that i) augments assembly rate by 3-fold upon binding to specific receptor of interest, ii) stabilizes ligand-receptor complex compared to both monomeric ligand and ligand aggregates, iii) triggers enhancement of pro-inflammatory cytokine release, cell viability and phosphorylation of transcription factor compared to binding-only molecule. Finally, the ligand also supplements anti-PD-1 therapy alone in CT26 syngeneic murine tumor model with controllable toxicity risk.