Hierarchical protein nano-crystalline hydrogel with extracellular vesicles for ectopic lymphoid structure formation

Abstract

Among cancer therapies, immune checkpoint blockade (ICB) has emerged as a prominent approach, substantially enhancing anti-tumor immune responses. However, the efficacy of ICB is often limited in the absence of a pre-existing immune response within the tumor microenvironment. Here, we introduce a novel hierarchical protein hydrogel platform designed to facilitate the formation of artificial tertiary lymphoid structures (aTLS), thereby improving ICB efficacy. Through the integration of self-assembling ferritin protein nanocages, rec1-resilin protein, and CP05 peptide, our hierarchical hydrogels provide a structurally supportive and functionally adaptive scaffold capable of on-demand self-repair in response to mild thermal treatments. The effective encapsulation of extracellular vesicles (EVs) via the CP05 peptide ensures the formation of aTLS with germinal center-like structures within the hierarchical hydrogel. We demonstrate that, combined with ICB therapy, EV-loaded hierarchical hydrogels also induce the TLS within the tumor, markedly promoting immune responses against ICB-resistant tumor. This bioactive hydrogel platform offers a versatile tool for enhancing a broad range of immunotherapies, with potential applications extending beyond TLS to other frameworks that support complex tissue architectures. • Protein nanocages serve as nanoscale building blocks, synergizing with elastomers to form a hierarchical macroscale scaffold. • The hydrogels exhibit on-demand thermal self-repair, preserving both structural integrity and functionality. • The hydrogelsencapsulate extracellular vesicles, forming artificial tertiary lymphoid structures to boost immunity. • Combined with immune checkpoint blockade (ICB), these hydrogels amplify immune activation and overcome ICB resistance.