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An in situ engineered chimeric IL-2 receptor potentiates the tumoricidal activity of proinflammatory CAR macrophages in renal cell carcinoma

Nature Cancer volume 6, pages 838–853 (2025) Cite this article

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Abstract

Chimeric antigen receptor macrophage (CAR-M) therapy has shown great promise in solid malignancies; however, the phenotypic re-domestication of CAR-Ms in the immunosuppressive tumor niche restricts their antitumor immunity. We here report an in situ engineered chimeric interleukin (IL)-2 signaling receptor (CSR) for controllably manipulating the proinflammatory phenotype of CAR-Ms, augmenting their sustained tumoricidal immunity. Specifically, our in-house-customized lipid nanoparticles efficiently introduce dual circular RNAs into macrophages to generate CSR-functionalized CAR-Ms. The intracellular inflammatory signaling pathway of CAR-Ms can be stimulated with the IL-2 therapeutic via the synthetic IL-2 receptor, which induces the antitumor phenotype shifting of CAR-Ms. Moreover, hydrogel-mediated combinatory treatment with lipid nanoparticles and IL-2 remodels the immunosuppressive tumor microenvironment and promotes tumor regression in renal carcinoma animal models. In summary, our findings establish that the proinflammatory phenotype of CAR-Ms can be modulated by a synthetic IL-2 receptor, benefiting the antitumor immunotherapy of CAR-Ms with broad application in other solid malignancies.

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Fig. 1: IL-2R–TLR4 CSR enables the activation of signaling pathways downstream of TLR4 by exogenous IL-2.
The alternative text for this image may have been generated using AI.
Fig. 2: Design and characterization of the LNP hydrogel delivery system.
The alternative text for this image may have been generated using AI.
Fig. 3: IL-2R–TLR4 CSR and anti-CA9 CAR expression and effector activity in vitro.
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Fig. 4: In vivo antitumor efficacy of injecting IL-2 and circRNA-mLNP@gel into the subrenal capsule.
The alternative text for this image may have been generated using AI.
Fig. 5: Macrophage polarization and phagocytosis activity in the tumor microenvironment following IL-2 and circRNA-mLNP@gel injection.
The alternative text for this image may have been generated using AI.
Fig. 6: In vivo antitumor efficacy of postoperative injection of IL-2 and circRNA-mLNP@gel into the tumor cavity.
The alternative text for this image may have been generated using AI.
Fig. 7: Therapeutic efficacy of IL-2 and circRNA-mLNP@gel in the hu-HSC-PDX RCC model and schematic illustration of the treatment strategy.
The alternative text for this image may have been generated using AI.

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Data availability

All data supporting the findings of this study are available within the article and its Supplementary Information files and from the corresponding authors upon reasonable request. RNA-seq data generated in this study have been deposited in the Genome Sequence Archive database at https://bigd.big.ac.cn/gsa/browse/CRA015275. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Key Research and Development Program of China (2023YFC2812400, W.J.; 2024YFA0918400, X.Y.J.), the National Natural Science Foundation of China (82350125 and 82173763, X.Y.J.), the ISF-NSFC Joint Scientific Research Program (52161145501, X.Y.J.), the Natural Science Foundation of Shandong Province (ZR2024YQ053, W.J.) and Taishan Scholar Program of Shandong Province (tsqnz20221166, W.J.). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. Several illustrations for this manuscript were created with BioRender.com.

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Author notes

  1. These authors contributed equally: Weiqiang Jing, Maosen Han, Ganyu Wang.

Authors and Affiliations

  1. Department of Urology, Qilu Hospital, Cheeloo College of Medicine; Shandong Key Laboratory of Targeted Drug Delivery and Advanced Pharmaceutics, NMPA Key Laboratory for Technology Research and Evaluation of Drug Products and Key Laboratory of Chemical Biology (Ministry of Education), Department of Pharmaceutics, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, China

    Weiqiang Jing, Maosen Han, Ganyu Wang, Zhichao Kong, Xiaotian Zhao, Zhipeng Fu, Xuewen Jiang, Chongdeng Shi, Chen Chen, Jing Zhang, Zuolin Zheng, Jinxin Gao, Weiyi Sun, Chunwei Tang, Zhenmei Yang, Yan Wang, Ying Liu, Kun Zhao, Benkang Shi & Xinyi Jiang

  2. Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Kowloon, China

    Danqing Zhu

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Contributions

X.Y.J., B.S., W.J. and M.H. conceived the study and designed experiments. M.H., Z.K. and Z.F. characterized the properties of the materials and performed the cell experiments. W.J., G.W., X.Z. and C.S. performed the animal experiments. X.W.J., C.C., J.Z., Z.Z., J.G., W.S., C.T., Z.Y., Y.W. and Y.L. contributed to data analysis and interpretation. W.J., M.H. and G.W. crafted the figures and wrote the manuscript. X.Y.J., B.S., D.Z., K.Z., W.J., M.H. and G.W. edited and revised the manuscript and supervised the research. The final draft of the manuscript was approved by all co-authors.

Corresponding authors

Correspondence to Weiqiang Jing, Benkang Shi or Xinyi Jiang.

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The authors declare no competing interests.

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Nature Cancer thanks Nico Lachmann, Lili Yang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Expression of CA9 and CD68 in patients with RCC.

(a) CA9 expression in kidney cancer tumor tissues (n = 531 tissues, biological replicates) and normal tissues (n = 100 tissues, biological replicates) based on the TCGA and GTEx databases. P = 2.4e-50 P values were calculated using Wilcoxon rank sum test. The middle line in the box represents the median, while the lower and upper edges of the box correspond to the first and third quartiles. The whiskers stretch to 1.5 times the interquartile range from the lower and upper quartiles. (b) Survival curves of kidney cancer patients with high (n = 701 patients, biological replicates) and low (n = 176 patients, biological replicates) expression of CA9. P values were calculated using Log-rank test P = 2.2e-7 (c) CD68 expression in kidney cancer tumor tissues (n = 531 tissues, biological replicates) and normal tissues (n = 100 tissues, biological replicates) based on the TCGA and GTEx databases. P = 5.5e-44 P values were calculated using Wilcoxon rank sum test. The middle line in the box represents the median, while the lower and upper edges of the box correspond to the first and third quartiles. The whiskers stretch to 1.5 times the interquartile range from the lower and upper quartiles. (d) Survival curves of kidney cancer patients with high (n = 635 patients, biological replicates) and low (n = 242 patients, biological replicates) expression of CD68. n is the number of biologically independent samples. P values were calculated using Log-rank test P = 1.1e-5 (e) H&E and immunohistochemical staining of CA9 and CD68 in kidney cancer tumor tissues and nearby normal tissues. Scale bar = 100 μm. (A) and (C) were generated using RSEM with UCSC TOIL RNA-seq recompute and these data can be accessed and downloaded from the UCSC XENA platform at the following link https://xenabrowser.net/datapages/?cohort=TCGA%20TARGET%20GTEx. (B) and (D) were sourced from The Human Protein Atlas version 22.0 and Ensembl version 109. These datasets are accessible for download with the following link https://v22.proteinatlas.org/about/download.

Extended Data Fig. 2 Characterization of ionizable lipids, transfection efficiency, and hydrogel formation.

(a) The synthetic route to NMS-C9H19. Using citric acid as an ionizable lipid linker, the NMS-C9H19 is obtained through a simple reaction with the N1,N1-dinonylethane-1,2-diamine. (b) 1H-NMR and 13C-NMR Spectra confirming the structure of NMS-C9H19. (c) Representative flow cytometry and quantitative analyses of the transfection effects of formulations 1-16 in BMDMs using flow cytometry. (The Best formulation result was shown in Fig. 2d.) Data are shown as mean ± s.d. (n = 4 BMDM samples per group, biological replicates). (d) Flow cytometry analysis (Representative pictures from 3 cell samples, biological replicates) of Cy5-circRNA uptake by macrophages following the use of mannose as a targeting ligand. Characterization of gelatin (Gel) and hyaluronic acid (HA) derivatives. 1H-NMR (e) and FT-IR (f) spectra of HA-CHO and Gel-CDH. (G) 1H-NMR spectra of HA-Gel.

Source data

Extended Data Fig. 3 Safety and immunogenicity assessment of circRNA-mLNPs.

(a–d) Cytotoxicity evaluation of CAR-mLNP (a, b) and CSR-mLNP (c, d) in BMDMs and HK-2 cells using the CCK-8 assay. Data are shown in mean ± s.d. (n = 6 cell samples per group, biological replicates). (e-h) Cytotoxicity evaluation of CAR-mLNP (e, f) and CSR-mLNP (g, h) in BMDMs and HK-2 cells using the LDH release assay. Data are shown in mean ± s.d. (n = 6 cell samples per group, biological replicates). (i, j) Apoptosis analysis of CAR-mLNP and CSR-mLNP in BMDMs and HK-2 cells. Data are shown in mean ± s.d. (n = 4 cell samples per group, biological replicates). (k, l) Macrophage polarization analysis of empty LNPs in BMDMs. Data are shown in mean ± s.d. (n = 4 cell samples per group, biological replicates). Data are analyzed with one-way ANOVA. ****p < 0.0001. G5, Group 5 treated with IL-2, CAR-mLNPs and CSR-mLNPs.

Source data

Extended Data Fig. 4 Expression and effector activity of IL-2R/TLR4 CSR and anti-CA9 CAR in human and murine macrophages.

(a) Quantitative flow cytometry analysis of double-positive BMDMs (CAR-Myc + CSR-His+) for CAR-Myc and CSR-His following treatment with either free circRNA or circRNA-mLNPs. Data are presented as mean ± s.d. (n = 4 cell samples per group, biological replicates) and analyzed using one-way ANOVA. ****p < 0.0001. (b) Quantitative flow cytometry analysis of PE-CSR-His in human primary macrophages after treatment with free circRNA or CSR-mLNPs. Data are shown as the mean ± s.d. (n = 4 cell samples per group, biological replicates). Data are analyzed with one-way ANOVA. ****p < 0.0001.(c) Western blot analysis of protein levels associated with innate immune signaling pathways (TAK1, p-TAK1, IKKβ, p-IKKα/β, p65, p-p65, JNK, p-JNK, ERK1/2, p-ERK1/2, p38 and p-p38) in CSR-expressing or untreated human primary macrophages stimulated with IL-2 (NPs: CSR circRNA compressed by mLNPs). (d) Heatmap depicting differentially expressed biomarkers in BMDMs following treatment with PBS or IL-2 + CSR-mLNP + CAR-mLNP, as analyzed through bulk-RNA sequence (n = 3 cell samples per group, biological replicates). (e) Western Blot (from 3 independent experiments) show the construction of the Renca cell line stably expressing human CA9. (f) The cytokines (including IL-12, TNF-α and IFN-γ) secreted by BMDMs after treatment with different formulations. Data are shown as mean ± s.d. (n = 4 cell samples per group, biological replicates). Data are analyzed with one-way ANOVA. *P = 0.0362 for G5 versus G4 and *P = 0.0105 for G3 versus G2 in IL-12 concentration, ***P = 0.0007 for G5 versus G4 in TNF-α concentration, ***P = 0.0003 for G5 versus G4 and *P = 0.0479 for G3 versus G2 in IFN-γ concentration, ****P < 0.0001. G1, Group 1 treated with PBS (control); G2, Group 2 treated with IL-2; G3, Group 3 treated with IL-2 and CAR-mLNP; G4, Group 4 treated with IL-2 and CSR-mLNP; and G5, Group 5 treated with IL-2, CAR-mLNP and CSR-mLNP.

Source data

Extended Data Fig. 5 In vivo analysis of the percentages and functions of tumor-infiltrating CD8+ and CD4 + T cells.

(a) Representative flow cytometry analysis showing the percentages of CD3 + CD8+ cells in tumor tissues from mice treated with different formulations. (b) Representative flow cytometry analysis of the percentages of Granzyme B+ cells among CD8 + T cells in tumor tissues from mice treated with different formulations. (c) Representative flow cytometry analysis of the percentages of CD3 + CD4+ cells in tumor tissues from mice treated with different formulations. (d) Representative flow cytometry analysis of the percentages of IFN-γ+ cells among CD4 + T cells in tumor tissues from mice treated with different formulations. (e) Representative flow cytometry analysis of the percentages of Foxp3+ cells among CD4 + T cells in tumor tissues from mice treated with different formulations. The results of A–E are expressed as the mean ± s.d (n = 4 tumor tissues per group, biological replicates). Statistical significance was calculated via one-way ANOVA. ***P = 0.0001 for G5 versus G4 in (C), **P = 0.003 for G3 versus G2 in (D), *P = 0.021 for G5 versus G4 in (E), ****p < 0.0001. G1, Group 1 treated with PBS (control); G2, Group 2 treated with IL-2@gel; G3, Group 3 treated with CAR-mLNP/IL-2@gel; G4, Group 4 treated with CSR-mLNP/IL-2@gel; and G5, Group 5 treated with CAR-mLNP + CSR-mLNP/IL-2@gel.

Source data

Extended Data Fig. 6 Kinetic analysis of CAR and CSR expression and the macrophage phenotype in tumors following treatment.

(a) Flow cytometry analysis and quantification of CAR expression in tumor-infiltrating macrophages at various time points after treatment with CAR-mLNP + CSR-mLNP/IL-2@gel. (b) Flow cytometry analysis and quantification of CSR expression in tumor-infiltrating macrophages at various time points after treatment with CAR-mLNP + CSR-mLNP/IL-2@gel. (c) Flow cytometry analysis and quantification of the percentage of M1 macrophages (CD80 + F4/80+) among tumor-infiltrating macrophages at various time points following treatment with CAR-mLNP + CSR-mLNP/IL-2@gel. The data of A–C are shown in mean ± s.d (n = 4 tumor tissues per group, biological replicates).

Source data

Extended Data Fig. 7 Effect of treatments on lung metastasis and kidney tumor in renal cancer mouse model.

(a) Representative photographs of the lungs of mice with renal cancer that were treated as indicated (top). Scale bar = 1 cm. Representative histopathological images of lung metastasis after H&E staining (middle), Scale bar = 1 mm. Photos of the lungs after fixation with picric acid to visualize pulmonary nodules (bottom), Scale bar = 1 cm. Representative pictures from n = 3 independent replicates. (b) Lung weights of kidney tumor-bearing mice after different treatments. (c) The numbers of lung nodules were counted. G1, Group 1 treated with PBS (control); G2, Group 2 treated with IL-2@gel; G3, Group 3 treated with CAR-mLNP/IL-2@gel; G4, Group 4 treated with CSR-mLNP/IL-2@gel; and G5, Group 5 treated with CAR-mLNP + CSR-mLNP/IL-2@gel; Normal, Group derived from non-tumor-bearing mice. (d) Representative photographs (scale bars, 1 cm) and H&E staining of tumor-bearing kidneys (scale bars, 500 μm) and lungs (scale bars, 1 mm) from different treatment groups. Representative pictures from n = 3 independent replicates. (e) Weights of tumor-bearing kidneys in mice after different treatments. (f) Weights of lungs in kidney tumor-bearing mice after different treatments. The data of B,C,E and F are presented as mean ± s.d. (n = 6 mice per group, biological replicates). Data are analyzed with one-way ANOVA. *P = 0.0215 for G5 versus G4 in (c), ***P = 0.0003 for CAR-mLNP + CSR-mLNP/IL-2@gel versus CAR-mLNP + CSR-mLNP/IL-2 in (e), ***P = 0.0003 for CAR-mLNP + CSR-mLNP/IL-2@gel versus CAR-mLNP + CSR-mLNP/IL-2 in (f), ns: no significance, ****p < 0.0001.(g) LPS detection in each component of CAR-mLNP + CSR-mLNP/IL-2@gel. The top part of the image presents the results of the diluted LPS standard, while the bottom part illustrates the results for the six components or their degradation products. Data are presented as mean ± s.d. (Representative pictures from 3 independent replicates) and analyzed using one-way ANOVA. ***p < 0.001, ****p < 0.0001.

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Extended Data Fig. 8 Impact of macrophage depletion or T cell deficiency on the antitumor efficacy of CAR-mLNP + CSR-mLNP/IL-2@gel.

(a) Representative histogram and (b) quantification of CD11b + F4/80+ macrophages in tumor tissues following different treatments. Control: treated with Control Liposomes (0.2 ml per mouse, administered via tail vein injection every three days); Macrophage depletion: treated with Clodronate Liposomes (0.2 ml per mouse, administered via tail vein injection every three days); G5: treated with CAR-mLNP + CSR-mLNP/IL-2@gel. Data are presented as mean ± s.d. (n = 4 tumor tissues per group, biological replicates). Data are analyzed with one-way ANOVA. ****p < 0.0001. (c) Representative photographs (scale bars, 1 cm) and H&E staining of tumor-bearing kidneys (scale bars, 500 μm) and lungs (scale bars, 1 mm) from different treatment groups. Representative pictures from n = 3 mice per group, independent replicates. Weights of tumor-bearing kidneys (d) and lungs (e) from mice after different treatments. Data are presented as mean ± s.d. (n = 6 mice per group, biological replicates) and analyzed using one-way ANOVA. ****p < 0.0001. ns, no significance. (f) Representative photographs (scale bars, 1 cm) and H&E staining of tumor-bearing kidneys (scale bars, 500 μm) and lungs (scale bars, 1 mm) from different groups. Representative pictures from n = 3 mice per group, independent replicates. Nude mice, which are deficient in adaptive immunity due to the lack of T cells but retain functional macrophages; G5: treated with CAR-mLNP + CSR-mLNP/IL-2@gel. Weights of tumor-bearing kidneys (g) and lungs (h) from mice in different groups. The data are presented as the mean ± s.d. (n = 6 mice per group, biological replicates) and were analyzed via one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. **P = 0.0054 for nude mice versus BALB/c in (G), ***P = 0.0002 for nude mice + G5 versus BALB/c + G5, *P = 0.0217 for nude mice versus BALB/c in (H).

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Extended Data Fig. 9 Edited macrophage specificity and functionality and their potential applications in various diseases.

(a–e) Flow cytometry analysis and quantification of CAR macrophages (CAR-Myc + F4/80+) in the livers (a), spleens (b), lungs (c), lymph nodes (d) and blood (e) of tumor-bearing mice treated with G5 (CAR-mLNP + CSR-mLNP/IL-2@gel). Data are presented as mean ± s.d. (n = 4 mice per group, biological replicates) and analyzed using Two-tailed Student’s t-test. ns, no significance. (f) Western blot analysis of Myc-CAR expression in T cells, B cells, neutrophils, macrophages, and tumor cells isolated from tumor tissue one week after subcapsular renal administration. G1: Group 1 treated with PBS (control); G5: Group 5 treated with CAR-mLNP + CSR-mLNP/IL-2@gel. Representative pictures from n = 3 cell samples per group, independent replicates. (g) Flow cytometry analysis of the phagocytosis of GFP+ Renca cells by BMDMs treated with different formulations. Data are presented as mean ± s.d. (n = 4 mice per group, biological replicates). (h) Flow cytometry analysis of the phagocytosis of Calcein-labeled primary renal tubular cells by BMDMs treated with different formulations. (g, h) Control, group treated with PBS. Data are presented as mean ± s.d. (n = 4 mice per group, biological replicates) and analyzed using Two-tailed Student’s t-test. ns, no significance; *P = 0.0137 for IL-2 + CAR-mLNP + CSR-mLNP versus Control in (g) ns, no significance. (i) Schematic representation of the experimental design. (j) Representative flow cytometry analysis quantifying CSR-His+ macrophages in hCA9+ Renca liver metastasis tumor tissues following CSR-mLNP/IL-2@gel treatment. (k) Schematic representation of the experimental design. (l) Representative flow cytometry analysis quantifying CSR-His+ macrophages in GL261 postoperative tumor tissues following CSR-mLNP/IL-2@gel treatment. The data of J and L are shown in mean ± s.d. (n = 4 mice per group, biological replicates). Data are analyzed with the Two-tailed Student’s t-test. ****p < 0.0001.

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Extended Data Fig. 10 In vivo safety assessment of circRNA-mLNPs and hydrogel.

(a) Blood routine evaluation in mice subjected to different treatment groups. (b) Blood biochemistry analysis, including liver function, kidney function, and cardiac injury markers, in mice subjected to different treatment groups. (c) Body weight monitoring across treatment groups. (d) H&E staining of major organs (scale bar, 200 μm) isolated from mice subjected to different treatment groups (left); H&E, Masson, and PAS staining of kidneys (scale bar, 100 μm) in each treatment group (right). Representative pictures from n = 3 mice per group, biological replicates. The data of A, B and C are shown in mean ± s.d. (n = 4 mice per group, biological replicates). G1, Group 1 treated with PBS (control); G2, Group 2 treated with IL-2@gel; G3, Group 3 treated with CAR-mLNP/IL-2@gel; G4, Group 4 treated with CSR-mLNP/IL-2@gel; and G5, Group 5 treated with CAR-mLNP + CSR-mLNP/IL-2@gel.

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Molar ratios of the LNP formulations in the library.

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The phagocytosis of engineered murine BMDMs (red) against tumor cells (Renca, green). The phagocytosis of IL-2+CAR-mLNP+CSR-mLNP treated macrophages against tumor cells. In brief, BMDMs (red) were treated with CAR-mLNP+CSR-mLNP and stimulated with IL-2. Then, the tumor cells (hCA9+Renca, green) were co-incubated (at a ratio of BMDMs to Renca cells at 1:1). Images were taken every 2 min to evaluate the CSR+CAR-mediated phagocytosis of BMDMs.

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Jing, W., Han, M., Wang, G. et al. An in situ engineered chimeric IL-2 receptor potentiates the tumoricidal activity of proinflammatory CAR macrophages in renal cell carcinoma. Nat Cancer 6, 838–853 (2025). https://doi.org/10.1038/s43018-025-00950-1

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