Abstract
Lysosome-targeting chimeras (LYTACs) represent a revolutionary targeted protein degradation technology. However, the advancement of LYTACs faces substantial challenges due to the limited diversity of lysosome-trafficking receptors. In this study, we identified folate receptor α (FRα) as a new class of lysosome-trafficking receptors capable of facilitating the degradation of membrane proteins. Leveraging a polyvalent crosslinking strategy, we developed FRα-targeting chimeras (FRTACs), including epidermal growth factor receptor-targeting FR-Ctx and PD-L1-targeting FR-Atz. The optimized FRTACs demonstrated subnanomolar potency in eliminating cell-surface targets, with efficacy dependent on both FRα expression and lysosomal activity. Specifically, FR-Ctx inhibited cancer cell proliferation, while FR-Atz enhanced T cell-mediated cytotoxicity against tumor cells. FR-Atz exhibited robust PD-L1 degradation efficiency in vivo and elicited tumor-specific immune responses by reprogramming the tumor microenvironment from an immunosuppressive to an immunostimulatory state in both RM-1 and humanized B16F10 mouse models. These findings establish FRTACs as a promising platform for the design of tumor-targeting LYTACs.
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Data availability
All data generated or analyzed during this study are included in this published article (and its Supplementary Information files). The flow cytometry gating strategy is provided in the Supplementary Information. Raw mass spectrometry data are available via ProteomeXchange with identifiers PXD058523, PXD058514 and PXD058539. Source data are provided with this paper.
Change history
26 June 2025
A Correction to this paper has been published: https://doi.org/10.1038/s41589-025-01976-3
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Acknowledgements
This study was supported by grants from the National Natural Science Foundation of China (grant nos. 82373777, 82273854, 82273960 and 81973366), the Beijing Nova Program (grant no. 20220484116) and the CAMS Innovation Fund for Medical Sciences (grant no. 2021-I2M-1-070). The funding sources did not participate in study’s design, data collection, data analysis, data interpretation or the preparation and submission of the manuscript. We thank Q. Wang in the State Key Laboratory of Natural and Biomimetic Drugs, Peking University for the experimental assistance of SPR technology. We thank scientists at DeepKinase Co., Ltd for experimental assistance with proteomic analysis.
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S.L., W.Z., H.D. and X.Z. conceived and designed this study. D.X. and F.X. synthesized the FRTACs and performed binding affinity assay. J.D. conducted western blot, confocal microscopy, in vivo mice experiments and data analysis. C.S., J.W. and Y.W. performed T cell killing. D.X., X.F., X.X. and B.T. performed flow cytometry and liquid chromatography with MS/MS. X.Z. and H.D. supervised the study, interpreted results, wrote and revised the manuscript.
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Extended data
Extended Data Fig. 1 FRTACs engage lysosome-trafficking folate receptor for high-efficient protein degradation.
Our work shows FRα is a new generation of LTR engaged in FRTAC for highly-efficient degradation of membrane proteins by polyvalent conjugating with folate molecules. Previous works utilize FRα as tumor-targeting receptor for drug delivery by one-to-one conjugating FRα-targeting agents.
Extended Data Fig. 2 HIC, UV-Vis spectroscopies and binding affinities of FRTACs.
a, Synthetic scheme for antibody-folate conjugates (FRTACs). b, 1H NMR spectrum of the key intermediate folate-PEG1000-NHS (400 MHz, DMSO). c,d, The conjugation reaction process and FAR of FR-Ctx, FR-Atz (c) and FR-Stz, FR-Ttz (d) were analyzed by HIC and UV-Vis spectroscopy. e, SPR assay analyzing of the affinity of Ctx binding to EGFR. f, SPR assay analyzing of the affinity of FAR-Atz (FAR 15) binding to FRα. g, SPR assay analyzing the affinity of FAR-Atz (FAR 15) binding to PD-L1. h, SPR assay analyzing the affinity of Atz binding to PD-L1. HIC and UV-Vis spectroscopy (c and d), SPR results (e-h) are representative of at least two independent experiments.
Extended Data Fig. 3 FRTACs hijack the FRα for targeted protein degradation.
a, Western blot analysis of total TROP2 levels in SKOV3 cells after 48-h treatment with indicated FR-Stz (FAR 20) or FR-Stz (FAR 2). b, Western blot analysis of total PD-L1 levels in HCC827 cells after 48-h treatment with indicated FR-Atz (FAR 15) or FR-Atz (FAR 2). c, Western blot analysis of total EGFR levels in SKOV3 cells after 48-h treatment with indicated FR-Ctx (FAR 2) or 50 nM Ctx. Quantifications of EGFR to Tubulin were shown on the right. d, Live-cell confocal microscopy images of H292 cells treated with 10 nM FITC-labeled Atz or FR-Atz (FAR 15) at 37 °C for 4 h, then labeled with LysoTracker red for 30 min. Scale bar, 30 μm. e, Live-cell confocal microscopy images of SKOV3 cells treated with 50 nM rabbit IgG-647 and 25 nM goat anti-rabbit IgG, or goat anti-rabbit FR-IgG, then labeled with LysoTracker green for 30 min. Scale bar, 30 μm. Data in a-c represent three independent experiments and are shown as mean ± s.e.m. Images (d and e) are representative of three independent experiments.
Extended Data Fig. 4 FRTACs promote the membrane protein degradation in cancer cells.
a, Western blot analysis of total EGFR levels in SKOV3 cells after treatment with 10 nM FR-Ctx (FAR 15) for the indicated time points. Quantifications of EGFR to Tubulin were shown on the right. b, Western blot analysis of total PD-L1 levels in H292 cells after treatment with 10 nM FR-Atz (FAR 15) for the indicated time points. Quantifications of PD-L1 to GAPDH were shown on the right. c,d, Western blot analysis of total TROP2 levels in SKOV3 cells after 48-h treatment with indicated FR-Stz (FAR 20, c) or treatment with 10 nM FR-Stz (FAR 20) for the indicated time points (d). Quantifications of TROP2 to Vinculin were shown on the bottom or right. e,f, HER2 levels in SKOV3 cells were examined by western blot after 48-h treatment with indicated FR-Ttz (FAR 15, e) or with 10 nM FR-Ttz (FAR 15) for the indicated time points(f). Quantifications of HER2 to Tubulin were shown on the bottom or right. g, Flow cytometry detecting the degradation of cell surface PD-L1 in HCC827 cells treated with or indicated concentrations of Atz or FR-Atz (FAR 15) for 48 h. The statistic of MFI of PD-L1 was shown on the right. Data in a-g represent three independent experiments, and shown as mean ± s.e.m.
Extended Data Fig. 5 FRTAC induces a lysosomal-and FRα-dependent targets degradation.
a, Confocal microscopy visualization and quantification of LysoTracker in HCC827 cells treated with 50 nM Atz or 10 nM FR-Atz (FAR 15) for 48 h or 1 mM LLOMe for 1 h. Scale bar, 30 μm. b, Western blot analysis of the expression levels of FRα, FRβ, EGFR or PD-L1 in different cancer cells. c, Western blot determination of the EGFR levels in SKOV3 and Hep3B cells treated with indicated concentration of FR-Ctx (FAR 15). d, Western blot analysis of the effect of FR-Ctx (FAR 15) on EGFR degradation in H292 cells transfected with siRNA against FRα or control. e, Western blot examination of the effect of FR-Ctx (FAR 15) on EGFR degradation in Hep3B cells transfected with increasing amounts of Myc-FRα (0.25, 0.5, 1 μg). f, Quantitative proteomics analysis of the changes of whole-cell H292 proteins after 48-h treatment with 10 nM FR-Atz (FAR 15) or 50 nM Atz shows no FRα degradation. Images (a) and data in b-e represent three independent experiments, data in f are the mean of three biological replicates. Data in a are shown as mean ± s.e.m. P values were calculated using an unpaired two-tailed t-tests.
Extended Data Fig. 6 FRTAC-mediated target degradation alleviates tumor cell growth and enhances the T cell-mediated tumor cell killing.
a, Cell impedance assay analysis of the proliferation of H292 cells treated with 50 nM Ctx or 10 nM FR-Ctx (FAR 15). b, Flow cytometry analysis of PBMCs-mediated killing of HCC827 cells treated with PBS, 50 nM Atz or 10 nM FR-Atz (FAR 15) using Annexin V and PI double staining. c,d PBS, 50 nM Atz, or 10 nM FR-Atz (FAR 15)-treatment HCC827 cells were co-incubated with activated PBMCs, the levels of IFN-γ (c) and GzmB (d) in CD8+T cells were examined by flow cytometry. Data in a–d represent three independent experiments, and data are shown as mean ± s.e.m. P values were calculated using an unpaired two-tailed t-tests.
Extended Data Fig. 7 PD-L1 degradation Effect of FR-Atz on mice bearing RM-1 tumors and B16-hPD-L1 cells.
a, Western blot analyzing the FRα and PD-L1 levels in mouse Lewis, RM-1, 4T1 and MC38 cells. b, Western blot determining the PD-L1 level in Lewis and RM-1 cells treated with PBS, 50 nM Atz or 10 nM FR-Atz (FAR 15) for 48 h. c, C57BL/6 mice bearing RM-1 tumor were intraperitoneal (i.p.) injected with PBS, Atz (5 mg/kg), Atz (5 mg/kg) + FA-PEG, FR-IgG (5 mg/kg) or FR-Atz (FAR 15, 5 mg/kg), the curves of the body weight were recorded every three days. n = 6 mice per group. NS, not significant. d, Heart, liver, kidney and spleen of C57BL/6 mice bearing RM-1 tumor treated with PBS, Atz (5 mg/kg) and FR-Atz (FAR 15, 5 mg/kg) were taken out for H&E staining. Scale bar, 30 μm. e, Western blot analyzing the PD-L1 level in B16-empty vector and B16-hPD-L1 cells. f, Western blot determining the PD-L1 level in B16-hPD-L1 cells treated with PBS, 50 nM Atz or 10 nM FR-Atz for 48 h. g, Western blot analysis of the PD-L1 abundance in spleen tissues from PBS, Atz, or FR-Atz (FAR 15)-treated nude mice bearing subcutaneous H292 tumor (n = 3). Data in a, b, d-g represent three independent experiments. Data in c were shown as mean ± s.e.m. P values were calculated using an unpaired two-way ANOVA test.
Extended Data Fig. 8 FR-Atz promotes antitumor immunity in mice bearing RM-1 tumor.
a-c, C57BL/6 mice bearing RM-1 tumor were intraperitoneal (i.p.) injected with PBS, Atz (5 mg/kg), Atz (5 mg/kg) + FA-PEG, FR-IgG (5 mg/kg) or FR-Atz (FAR 15, 5 mg/kg). Flow cytometry analyzing the populations of tumor-infiltrating CD8+ T cells (a), and effector molecules GzmB (b) and IFN-γ (c) in CD8+ T cells. (n = 6). d-f, Flow cytometry analyzing the populations of tumor-infiltrating Tregs (d), M-MDSCs and PMN-MDSCs (e), M1 and M2 macrophages (f), (n = 6). Data in a-f are shown as mean ± s.e.m. P values were determined by unpaired two-tailed t-test.
Supplementary information
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Xiao, D., Dong, J., Xie, F. et al. Polyvalent folate receptor-targeting chimeras for degradation of membrane proteins. Nat Chem Biol (2025). https://doi.org/10.1038/s41589-025-01924-1
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DOI: https://doi.org/10.1038/s41589-025-01924-1
