Drug tolerance is the basis for acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) including osimertinib, through mechanisms that still remain unclear. Here, we show that while AXL-low expressing EGFR mutated lung cancer (EGFRmut-LC) cells are more sensitive to osimertinib than AXL-high expressing EGFRmut-LC cells, a small population emerge osimertinib tolerance. The tolerance is mediated by the increased expression and phosphorylation of insulin-like growth factor-1 receptor (IGF-1R), caused by the induction of its transcription factor FOXA1. IGF-1R maintains association with EGFR and adaptor proteins, including Gab1 and IRS1, in the presence of osimertinib and restores the survival signal. In AXL-low-expressing EGFRmut-LC cell-derived xenograft and patient-derived xenograft models, transient IGF-1R inhibition combined with continuous osimertinib treatment could eradicate tumors and prevent regrowth even after the cessation of osimertinib. These results indicate that optimal inhibition of tolerant signals combined with osimertinib may dramatically improve the outcome of EGFRmut-LC.

Extracellular vesicles (EVs) are important mediators of cell–cell communication, including immune regulation. Despite the recent development of several EV‐based cancer immunotherapies, their clinical efficacy remains limited. Here, we created antigen‐presenting EVs to express peptide‐major histocompatibility complex (pMHC) class I, costimulatory molecule and IL‐2. This enabled the selective delivery of multiple immune modulators to antigen‐specific CD8 ⁺ T cells, promoting their expansion in vivo without severe adverse effects. Notably, antigen‐presenting EVs accumulated in the tumour microenvironment, increasing IFN‐γ ⁺ CD8 ⁺ T cell and decreasing exhausted CD8 ⁺ T cell numbers, suggesting that antigen‐presenting EVs transformed the ‘cold’ tumour microenvironment into a ‘hot’ one. Combination therapy with antigen‐presenting EVs and anti‐PD‐1 demonstrated enhanced anticancer immunity against established tumours. We successfully engineered humanized antigen‐presenting EVs, which selectively stimulated tumour antigen‐specific CD8 ⁺ T cells. In conclusion, engineering EVs to co‐express multiple immunomodulators represents a promising method for cancer immunotherapy.

Extracellular vesicles (EVs) are emerging as versatile nanocarriers for targeted drug delivery and immune modulation. However, strategies that can induce antigen-specific immune tolerance remain limited, highlighting an unmet need for more precise and effective approaches. To address this challenge, we aimed to develop a modular EV-based system capable of inducing antigen-specific regulatory T cells (Tregs). In this study, we developed engineered antigen-presenting EVs (AP-EVs) that co-display peptide-major histocompatibility complex class II complexes (pMHCII), interleukin-2 (IL-2), and transforming growth factor-β (TGF-β) on their surface. These immunomodulatory molecules were anchored to the EV membrane via CD81 or milk fat globule-EGF factor 8 (MFG-E8) scaffolds to ensure stable and multivalent presentation. AP-EVs induced the differentiation of antigen-specific Tregs from naïve CD4⁺ T cells in vitro, and promoted their proliferation and expression of canonical regulatory markers, including CD25, CTLA-4, PD-L1, and LAG-3. In vivo, the combination of AP-EVs and mTOR inhibition with rapamycin significantly enhanced the generation of Foxp3⁺ Tregs in antigen-specific adoptive transfer models. The Tregs induced by AP-EVs in vitro exhibited suppressive function, highlighting the therapeutic potential of this system. Our findings establish a modular, cell-free EV platform for antigen-specific immune tolerance, with potential applications in the treatment of autoimmune and allergic diseases through targeted immune regulation.

Extracellular vesicles (EVs), particularly small EVs (sEVs), are lipid bilayer vesicles secreted by various cell types and play a key role in intercellular communication. These vesicles are promising tools for cancer immunotherapy owing to their biocompatibility, low immunogenicity, and capacity for targeted drug delivery. In this study, we aimed to assess the potential of engineered antigen-presenting EVs (AP-EVs) to selectively expand and differentiate antigen-specific CD4⁺ T cells. We engineered two types of AP-EVs: AP-EVs-Th1 expressing MHC class II, CD80, and interleukin (IL)-12 on their surface to promote Th1 differentiation, and AP-EVs-Th2 expressing MHC class II, CD80, and IL-4 to induce Th2 differentiation. In vitro experiments demonstrated that AP-EVs successfully induced the antigen-specific proliferation and differentiation of Th1 and Th2 cells, respectively. Notably, in vivo administration of AP-EVs-Th1 significantly enhanced the proliferation and differentiation of tumor antigen-specific Th1 cells, leading to robust anti-tumor effects in a murine melanoma model. These findings highlight the potential of AP-EVs-Th1 for cancer immunotherapy, particularly in augmenting CD4⁺ T cell responses. Furthermore, the versatility and adaptability of EV-based therapies make them beneficial for the development of personalized immunotherapeutic strategies for various cancer types, offering the advantages of targeted immune modulation, ease of use, and reduced risk compared to cell-based therapies.