Improving CAR-T Cell Function through a Targeted Cytokine Delivery System Utilizing CAR Target-Modified Extracellular Vesicles

Introduction The therapeutic response of chimeric antigen receptor (CAR) T cells is challenged by poor T-cell persistence and sustained anti-tumor activity. Incorporating cytokine signals, such as interIeukin-12 (IL-12), has emerged as a promising strategy to enhance CAR-T cell functionality due to its potent immunostimulatory effects. However, clinical application of IL-12 is constrained by severe toxicity associated with systemic exposure. Therefore, alternative agents or delivery systems are desired to exploit the benefits of IL-12 safely. This study investigated the therapeutic potential of a novel IL-12 delivery system utilizing extracellular vesicles (EVs) modified with the target antigen CD19 to specifically deliver IL-12 to anti-CD19 CAR-T cells, thus augmenting their anti-tumor effects. Methods IL-12-anchored extracellular vesicles (IL-12 EVs) were generated from engineered HEK293T cells expressing IL-12 on the cell membrane via GPI structure. EVs with surface-displayed IL-12 and CD19 (CD19/IL-12 EVs) were produced from HEK293T cells co-expressing CD19 and IL-12. The physicochemical characteristics of the EVs were analyzed using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blot (WB). Binding and uptake efficiency of the EVs with CAR-T cells were validated using flow cytometry and imaging flow cytometry. In vitro experiments were conducted to investigate the effects of the EVs on the cytotoxic function, degranulation, cytokine secretion, and proliferation of CAR-T cells. Additionally, the therapeutic efficacy and safety of anti-CD19 CAR-T cells combined with intratumoral injection of CD19/IL-12 EVs were evaluated in a mouse model of Raji lymphoma xenograft. To explore underlying molecular mechanisms of CD19/IL-12 EVs, RNA-seq of CAR-T cells incubated with control EVs, CD19 EVs, IL-12 EVs or CD19/IL-12 EVs was performed. Results IL-12 on the surface of EVs was quantified by enzyme-linked immunosorbent assay. Co-culture of IL-12 EVs with anti-CD19 CAR-T cells significantly enhanced the effector function of CAR-T cells in vitro, resulting in increased secretion of IFN-γ and TNF-α, improved cytolytic activity, and enhanced expansion compared to equivalent concentration of soluble recombinant human IL-12 (rhIL-12). Compared to IL-12 EVs, CD19/IL-12 EVs exhibited superior binding efficiency to anti-CD19 CAR-T cells, but not to T cells, indicated by imaging flow cytometry. In a xenograft mice model bearing CD19+ Raji tumors, intratumoral injection of CD19/IL-12 EVs resulted in durable anti-tumor responses and enhanced in vivo expansion of CAR-T cells, outperforming the CD19 EVs, IL-12 EVs or control EVs, without causing systemic toxicity. RNA-seq analysis of CAR-T cells stimulated by CD19/IL-12 EVs suggest that the improved efficacy was driven by the IL-12 signaling. A total of 20 differentially expressed genes (16 upregulated genes and 4 downregulated genes) shared the same expression pattern of control EVs-versus-IL-12 EVs and CD19 EVs-versus-CD19/IL-12 EVs. These IL-12 related genes were demonstrated to be positively associated with favorable clinical response in patients with chronic lymphocytic leukemia receiving anti-CD19 CAR-T cell therapy. Conclusions This study provided the first line of evidence supporting the use of CAR target-modified EVs as a localized and targeted delivery system for the pleiotropic cytokine IL-12, serving as an effective and safe adjuvant in combination therapy with CAR-T cells.