To overcome these challenges, researchers have continuously upgraded and modified CAR-T cells. For example, incorporating different costimulatory signaling domains, such as CD28 or 4-1BB, addresses the short in vivo survival and poor activity of CARs. Simultaneously incorporating two costimulatory molecules into third-generation CARs enhances cytokine secretion. To better target the tumor microenvironment, researchers have developed fourth-generation CAR-T cells—"armored" T cells.
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There are three main types of "armored" CAR-T cells: universal cytokine-redirected CAR-T cells, cytokine-regulated CAR-T cells, and antibody-secreting CAR-T cells. Armored CAR-T cells release or modulate cytokines such as IL-12, IL-15, and IL-18, or deliver secreted antibodies directly to tumor sites. Consequently, they offer advantages such as enhanced cell infiltration, resistance to immunosuppression in the tumor microenvironment, and improved persistence, enhancing anti-tumor effects while reducing associated toxicity.
Several groundbreaking studies have demonstrated that T cells "armed" with cytokines such as IL-2, IL-12, IL-15, IL-18, Flt3L, IFNγ, and IL-7 have significant potential for treating solid tumors. However, peripheral expression of proinflammatory cytokines can lead to toxicity, a significant concern in the development of armored CAR-T cells.
Researchers have developed various strategies to restrict armor gene expression to tumor sites, such as the NFAT-inducible promoter and the synthetic Notch system. However, these strategies are limited to short regulatory DNA fragments and cannot achieve precise and site-specific regulation of gene expression. This has tragically led to severe toxicity in clinical trials of NFAT-IL-12 armored CAR-T cells.
Figure 1. Development of a CRISPR knock-in strategy to engineer armoured T cells with tumour-restricted transgene expression. (Chen A X Y, et al., 2025)
In this Nature paper, the research team utilized CRISPR gene editing technology to knock in transgenes into specific gene loci via homology-directed repair (HDR). For example, knocking in IL-12 into the NR4A2 or RGS16 promoter sites induced significant therapeutic responses and improved long-term survival in mice without any significant toxicity. Furthermore, the cured mice exhibited durable immunity against secondary tumors, demonstrating that these "armored" CAR-T cells can not only eliminate existing tumors but also establish long-lasting immune memory to prevent tumor recurrence.
Overall, by leveraging endogenous gene regulatory mechanisms to express proinflammatory cytokines in a tumor-localized manner, the team aims to expand the repertoire of cytokines that can be safely expressed by "armored" CAR-T cells, potentially overcoming the challenges of CAR-T cell therapy for solid tumors.
Reference
Chen A X Y, et al. Rewiring endogenous genes in CAR T cells for tumour-restricted payload delivery. Nature, 2025: 1-11.