Science Bulletin│Shanghai Institute of Materia Medica developed a tumor-targeted lipoplex to generate in situ NKG2D CAR T/NK cells to enhance tumor immunotherapy

Chimeric antigen receptor CAR-T cell therapy has achieved remarkable therapeutic effects in the treatment of hematological tumors, but the treatment of solid tumors still faces challenges. Traditional CAR-T cells need to be expanded in vitro and then reinfused, which requires complex processes, high costs, long cycles, and difficulty in effectively infiltrating solid tumors after infusion, resulting in limited efficacy. In recent years, the use of lipid nanocarriers to directly generate CAR cells in vivo has become a new research direction, but how to achieve "precise generation, local activation, safety and controllability" is still a big problem. Therefore, there is an urgent need to develop a new technology that can accurately target the generation of CAR-T/NK cells and have good safety.

In response to the above challenges, Li Yaping's team at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and Wang Dange's team at Shanghai Jiao Tong University published a research result titled "In situ generation of NKG2D CAR T/NK cells via a tumor-targeted lipoplex for enhancing cancer immunotherapy" in Science Bulletin on August 2, 2026. This study developed a lipid delivery system (R8L-NI@Lipo) that targets metabotropic glutamate receptor 4 (GRM4) and has tumor penetration capabilities, and achieves precise CAR compilation within tumor tissues by delivering hypoxia-responsive pDNA encoding NKG2D-CAR and IL-15 (Figure 1). R8L-NI@Lipo achieves deep tumor penetration through R8 penetration-enhancing peptide, uses LSOP molecules to target GRM4 receptors in tumor tissues to achieve tumor-specific enrichment, and simultaneously secretes IL-15 to achieve sustained stimulation of NK and CD8+ T cells. The pDNA loaded by the lipid delivery system contains 9×HRE hypoxia response element and ODD oxygen-dependent degradation domain, which ensures that CAR is only expressed in the tumor hypoxic microenvironment and reduces the non-specific expression of NKG2D-CAR in normal tissues. Research results show that in a variety of triple-negative breast cancer (TNBC) mouse models, NKG2D CAR-T/NK cells can be generated in situ in the tumor, reshape the tumor immune microenvironment, significantly inhibit tumor growth and extend survival, while effectively avoiding off-target toxicity in normal organs such as the liver.

Based on the high expression of GRM4 in breast cancer tissues and various immune cells (T cells, NK cells, etc.), the research team selected liposomes modified with the GRM4 agonist L-O-phosphoserine (LSOP) containing a phosphate group, and combined it with a guanidine group Octameric arginine (R8) was conjugated to form a LOSP-modified R8 peptide-coupled DSPE-PE distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG-R8L) carrier that targets GRM4 and can penetrate the tumor vascular barrier. This vector can efficiently compress plasmid pDNA (Dzr) carrying the piggyBac transposon system, which encodes NKG2D chimeric antigen receptor (CAR) and IL-15 and is regulated by a hypoxia response element (HRE) and an oxygen-dependent degradation domain (ODD). In vitro and in vivo experiments have confirmed that NKG2D CAR is significantly expressed only under hypoxic (≤1% O₂) conditions and is almost silent under normoxia, thus achieving the generation of CAR-T/NK cells specific to the tumor hypoxic microenvironment. Compared with the normoxic-regulated control plasmid (Cme), the Dzr plasmid delivered by R8L-NI@Lipo significantly reduced non-specific CAR expression in normal organs such as the heart, liver, spleen, lung, and kidney, and avoided toxicity such as leukopenia and abnormal liver function. At the same time, transcriptome analysis showed that this system specifically activated anti-tumor immune pathways such as antigen presentation, NK cytotoxicity, and the complement cascade (Figure 2). In subcutaneous 4T1 and EMT6 mouse models, R8L-NI@Lipo significantly increased the infiltration of NKG2D⁺CD8⁺ T cells and NK cells in tumors, increased the levels of effector molecules such as granzyme B and IFN-γ, induced the polarization of M1 macrophages and reduced regulatory T cells, reshaping the immune microenvironment. The tumor volume and weight of the treatment group decreased significantly (the average tumor weight in the 4T1 model was only 9.2% of that of the control group), the median survival period was extended from 22.5 days to 42.5 days (4T1) or 44.5 days (EMT6), and some mice achieved complete remission (Figure 3). This study developed a tumor-permeable lipid complex targeting GRM4, R8L-NI@Lipo, which can achieve hypoxia-responsive in situ generation of NKG2D CAR-T/NK cells in TNBC tissues, providing a new accurate, safe and efficient in situ generation strategy for CAR immunotherapy of solid tumors.

Researcher Li Yaping from Shanghai Institute of Materia Medica and Researcher Wang Dange from Shanghai Jiao Tong University are the co-corresponding authors of this article. Xu Xiaoxuan, a doctoral candidate at Shanghai Institute of Materia Medica, Yi Wenzhe, a postdoctoral fellow, and Sun Xiangshi, a doctoral candidate at Jilin University, are the co-first authors of this article. This research was funded by Lingang Laboratory scientific research tasks, major national science and technology projects and other projects.

Full text link: https://www.sciencedirect.com/science/article/pii/S2095927326008728

Figure 1 Construction of the lipid drug delivery system R8-NI@Lipo with tumor permeability and hypoxic microenvironment response and the principle of in situ production of CAR-T/NK inside solid tumors

Figure 2 (a) Difference in expression of GRM4 in human breast cancer (BRCA) and healthy tissues from TCGA; (b) Immunohistochemistry of GRM4 in patient breast tissue microarray (n=40); (c) t- SNE and GRM4 expression; (d) Preparation process of R8L-NI@Lipo; (e) Particle size and transmission electron microscope (TEM); (f) In vivo fluorescence imaging (λex/λem) of DiR-labeled R8L-NI@Lipo over time = 740/780 nm); (g) Extravasation image 24 h after injection, anti-CD31 antibody (green); (h, i) Construction of piggyBac transposon plasmid; (j) Flow cytometry detection of NKG2D CAR on spleen cells after 48 h of normoxia or hypoxia treatment (n=3); (k) ~100 or 200 Abundance of HIF-1α in mm³ tumors; (l) HIF-1α expression (n=3); (m) Co-localization map of tumor sections after R8L-NI@Lipo treatment: EGFP (white), HIF-1α (green), NKG2D (red), nucleus (blue); (n) Signal intensity within the depicted area; (o) PBS or R8L-NI@Lipo pretreatment 48 h later, the expression of NKG2D on splenocytes (n=3); (p) the expression of NKG2D in tumors; (q, r) the fold change of NKG2D CAR in CD8⁺ T cells (q) and NK cells (r) (n=3); (s) the level of IL-15 in tumors at 48 h (n=3).

Figure 3 (a–e) NKG2D on immune cells in heart (a), liver (b), spleen (c), lung (d) and kidney (e) Expression of CAR (n=4); (f) Staining of healthy organs: IL-15 (green), NKG2D (red), nucleus (blue); (g) White blood cell (WBC) density (n=5); (h) Alanine aminotransferase (ALT) level in serum (n=5); (i) Treatment and analysis timeline; PBS (G1), NI (G 2), NI@Lipo (G3), R8-NI@Lipo (G4), R8L-I@Lipo (G5), R8L-N@Lipo (G6) and R8L-NI@Lipo (G7), n=4 in each group; (j–l) Mechanism of immune activation by R8L-NI@Lipo: NK cells (j), GzmB⁺ NK cells (k) and IFN-γ⁺ NK cells (l); (m–p) Percentage of immune cells in tumors: CD8⁺ T cells (m), GzmB⁺ CD8⁺ T cells (n), IFN-γ⁺ CD8⁺ T cells (o) and regulatory T cells (Tregs) (p); (q) Tumor growth curve in 4T1 tumor model (n=6); (r) Tumor weight on day 15 (n=6); (s) Survival curve and median survival time (MST); (t) EMT6 tumor growth curve (n=6); (u) Body weight change curve (n=6); (v) Tumor survival curve and MST.

(Contributing department: Li Yaping’s research group; contributor: Yi Wenzhe; reviewer: Diao Wentong; editor: Song Wenke)