Intratumoural vaccination via checkpoint degradation-coupled antigen presentation

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Intratumoural vaccination via checkpoint degradation-coupled antigen presentation

Data availability

All data presented are available in the Article and its Supplementary Information. Raw RNA-seq data have been deposited in the Genome Sequence Archive of National Genome Data Center under accession identifier CRA031318. Raw proteomics data have been deposited in the ProteomeXchange consortium via the iProX partner repository under dataset identifier IPX0013811000. Source data are provided with this paper.

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Acknowledgements

We thank S. Liu for expert technical assistance; Y. Zhang, Y. Cao and S. Qin for discussions; and Z. Li and M. Li for their assistance in our OVA immunization. This work was funded by the Major Program of Shenzhen Bay Laboratory (S211101001 to P.R.C.); the National Key Research and Development Program of China (2016YFA0501500 to P.R.C.); the National Natural Science Foundation of China (grants 21740001, 21937001 and 22207076, 8200909770 to P.R.C., and grant 22477083 to H.Z.); the Beijing Natural Science Foundation (Z200010 to P.R.C.); the Tencent Foundation through the XPLORER PRIZE and the New Cornerstone Investigator Program (to P.R.C.); and the China National Postdoctoral Program for Innovation Talents (BX20220004 to Y.H.).

Author information

Author notes

  1. These authors contributed equally: Yu Han, Yicong Ma, Miao Pei

Authors and Affiliations

  1. Institute of Chemical Biology, Shenzhen Bay Laboratory, Shenzhen, China

    Yu Han, Miao Pei, Liyu Guo, Yike Fang, Chunjiang Deng, Su Zhao, Xueyin Lu, Heng Zhang & Peng R. Chen

  2. Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, China

    Yu Han, Yicong Ma, Weiming Guo & Peng R. Chen

  3. Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China

    Yu Han, Yicong Ma & Peng R. Chen

  4. Department of Biomedical Engineering, College of Future Technology, Peking University, Beijing, China

    Shenyi Yin, Jiahao Wang & Jianzhong Jeff Xi

  5. Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, China

    Weiming Guo & Peng R. Chen

Authors

  1. Yu Han
  2. Yicong Ma
  3. Miao Pei
  4. Shenyi Yin
  5. Jiahao Wang
  6. Liyu Guo
  7. Yike Fang
  8. Weiming Guo
  9. Chunjiang Deng
  10. Su Zhao
  11. Xueyin Lu
  12. Jianzhong Jeff Xi
  13. Heng Zhang
  14. Peng R. Chen

Contributions

P.R.C. and H.Z. conceptualized the idea and conceived the project. P.R.C., H.Z. and J.J.X. supervised the work. Y.H. and Y.M. developed the methods. M.P., S.Z. and X.L. optimized FnFSYs. Y.H. performed iVAC experiments with the help of Y.M. and W.G.; Y.M. performed analysis of RNA-seq and immunopeptidome data. C.D. and M.P. performed analysis of animal samples. Y.F. and L.G. performed CMV-associated assays. S.Y. and J.W. performed PTC-associated assays. Y.H. and Y.M. contributed to the writing of the original draft. P.R.C., H.Z. and J.J.X. reviewed the manuscript. All of the authors contributed to revising the manuscript and approved the final version.

Corresponding authors

Correspondence to Jianzhong Jeff Xi, Heng Zhang or Peng R. Chen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks Xin Zhou and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Development of optimized proximity-reactive FnFSYs for iVAC-mediated PD-L1 degradation-coupled antigen presentation.

a, Model reactions of mono- or di-fluoro phenyl sulfurofluoridates (FnPSFs) with imidazole informed optimization of FnFSYs. 1 mM FnPSF reacted with 100 mM imidazole under the same conditions, analysed by LC-MS (n = 3 independent replicates). b, Representative LC curves of model reactions after 4 h. c, Relative reactivity of FnPSF variants with imidazole compared to the original form (n = 3 independent replicates). d, Chemoenzymatic synthesis of FnFSYs: Tyrosine phenol lyase (TPL) enabled scalable production of fluoride-substituted tyrosines, creating two mono-fluoro and four di-fluoro FnFSYs through fluorosulfurylation. e, Crosslinking reactions between FnFSYs and imidazole: 1 mM FnFSY reacted with 100 mM imidazole in Tris buffer (pH = 8.0) at 37 °C for 4 h; conversion was analysed by LC-MS (n = 3 independent replicates). f, Representative LC curves of FnFSYs reacting with imidazole after 4 h. g, Relative reactivity of FnFSYs with imidazole compared to the original FSY (n = 3 independent replicates). Data are the mean ± s.d. Statistics: one-way ANOVA (c, g) with Tukey’s multiple comparisons test. NS: not significant.

Source data

Extended Data Fig. 2 Engineering bioorthogonal aminoacyl-tRNA synthetase for site-specific incorporation of FnFSYs into GFP.

a, Targeted saturation mutagenesis sites on PylRS. The chFSYRS V366A PylRS variant, identified after four rounds of positive selection, enables bio-orthogonal recognition of all FnFSYs. (PDB: 3QTC). b, Comparison of FnFSY incorporation into full-length GFP in E. coli by the chFSYRS V366A variant versus the original chFSYRS. PrUAA: proximity-reactive unnatural amino acids. Data are representative of three biological replicates. For gel source data, see Supplementary Fig. 1. c–e, Sequencing results of four targeted sites: (c) before selection, (d) after three rounds of positive selection, and (e) after a final round of positive selection. f,g, Quantitative analysis of EGFP expression by fluorescence: (f) Representative fluorescence imaging normalized by OD600, measured with a Typhoon FLA 9500 Fluorescence Imager. (g) Fluorescence intensity per group, measured by plate reader and normalized to FSY (n = 3 biological replicates). Data are the mean ± s.d. Statistics: two-way ANOVA (g) with Tukey’s multiple comparisons test.

Source data

Extended Data Fig. 3 The enhanced proximal crosslinking reactivity of iVAC accelerated PD-L1 degradation that promoted antigen presentation.

a, SDS-PAGE and quantitative analysis of proximal crosslinking efficiency of GlueBody variants with distinct FnFSYs to PD-L1. Purified proteins (5 μM) were incubated with PD-L1 (0.5 μM) in PBS buffer at 37 °C for 30 min (n = 3 biological replicates). b, Time-course analysis of PD-L1 degradation in HCC2935 cells treated with GlueBody-CPP or GlueBody2-CPP (n = 3 biological replicates). c, Enhanced antigen presentation on hPD-L1/MC38 cells mediated by iVAC with optimized GlueBody (n = 3 biological replicates). d, Targeted PD-L1 degradation and antigen presentation induced by iVAC on hPD-L1/MC38 cells. e,f, iVAC shows comparable effects on (e) PD-L1 degradation in HCC2935 cells and (f) T cell activity restoration in a PD-1/PD-L1 functional assay (n = 3 biological replicates). Data are the mean ± s.d. Statistics: one-way ANOVA (a) and two-way ANOVA (b, c, e, f), each followed by Tukey’s multiple comparisons test. NS: not significant.

Source data

Extended Data Fig. 4 The endogenous PD-L1 expression level is sufficient for iVAC-induced exogenous antigen delivery and presentation.

a, Relative expression levels of hPD-L1 on various tumour cell lines validated by flow cytometry. b,c, iVAC exhibited comparable target degradation and antigen presentation abilities on hPD-L1endo/MC38 cells to those on hPD-L1/MC38 cells after 6 h. For a–c, data were derived from three biological replicates. d, In the in vivo tumour inhibition experiment, hPD-L1endo/MC38 cells demonstrate similar performance to that of hPD-L1/MC38 cells with iVAC treatment. Following three immunizations with OVApep, 5 × 105 hPD-L1endo/MC38 cells with the endogenous hPD-L1 level were subcutaneously injected into the right flank of PD-L1 humanized C57BL/6J mice. When tumours reached 50–100 mm3, intratumoural administration of iVAC was given every three days for a total of four times (n = 7). Growth curves of hPD-L1endo/MC38 tumours treated with HBSS, OVApep, iVAC-CMV, CpG and iVAC-OVA (plotted on a Log2 scale). e, The activation ratio of CMV-specific JC5 cells following incubation with iVAC-CMV-treated wild-type (WT) MDA-MB-231 cells or hPD-L1/MDA-MB-231 cells that overexpress PD-L1 (n = 3 biological replicates). f, The endogenous expression and up-regulation of CMTM6 in human cancer cells did not attenuate hPD-L1 degradation triggered by iVAC. Data are representative of two biological replicates. For gel source data, see Supplementary Fig. 1. Data are the mean ± s.d. Statistics: two-way ANOVA (b,c,e) with Tukey’s multiple comparisons test. NS: not significant.

Source data

Extended Data Fig. 5 Evaluation of iVAC in PD-L1 humanized C57BL/6J mice.

a, Schematic overview of the biodistribution and plasma concentration study and treatment protocol. PD-L1 humanized mice were subcutaneously (s.c.) injected with 5 × 105 hPD-L1endo/MC38 cells in the right flank. When tumours reached 50–100 mm3, AF647-labelled drugs were administered intratumourally (i.t.) once. At different designated time points within 72 h, the fluorescence intensity in mouse serum (b) and the retention of fluorescence at the tumour site (c) were assessed (n = 4 mice per group). d, The biodistribution images of intratumourally injected drugs into hPD-L1endo/MC38 tumours 24 h post-administration (n = 4 mice per group). e, Tolerability of the iVAC in PD-L1 humanized C57BL/6J mice after OVApep immunization. Different doses of iVAC were injected subcutaneously (s.c.) to mice for a total of 6 times once every 3 days for tolerability evaluation. f, Mouse bodyweight was recorded during the treatment (n = 4 mice per group). g, On day 19, mice were euthanized, and organs were obtained and sliced for hematoxylin and eosin (H&E) staining and histology analysis. Scale bar: 500 µm. Data are representative of four mice per group.

Source data

Extended Data Fig. 6 APC-like tumour reprogramming induces an inflammatory profile in mouse cancer cells.

a, Confocal imaging of iVAC internalization in MDA-MB-231 cells using a FRET assay. Cy3 signal represents released OVA antigen; FRET signal indicates intact iVAC. Experiments were repeated independently two times. Scale bar: 10 μm. b, Following iVAC-induced antigenicity reprogramming, the leakage of delivered antigen from lysosomes for cross-presentation in tumours occurs via perforin-2 (Mpeg1). RNAi-mediated knockdown of perforin-2 can almost abrogate the antigen presentation by iVAC (n = 3 biological replicates). c–e, Heatmaps showing expression of genes related to (c) cell cycle, (d) IFN-γ and (e) STING pathways (n = 3 biological replicates). f, Gene set enrichment analysis (GSEA) for innate immune system genes, immune effector processes, and cytokine production in immune response genes in APC-like tumours derived from B16 cells compared to parental lines (HBSS, n = 3 biological replicates). Normalized enrichment score (NES) and false discovery rate q-values (FDR q) are provided. g,h, Quantitative PCR analysis of mRNA expression for antigen processing and presentation-related genes in APC-like tumours derived from (g) B16 and (h) MC38 cells (n = 3 biological replicates). In these experiments, the MC38 and B16 cells utilized were all murine PD-L1 knockout cells that expressed human PD-L1. Data are the mean ± s.d. Statistics: one-way ANOVA (b) and two-way ANOVA (g, h), each followed by Tukey’s multiple comparisons. NS: not significant.

Source data

Extended Data Fig. 7 The iVAC-converted APC-like tumour cells become immunogenic and elicit CD8+ T cell responses.

a,b, Expansion of OT-I memory T cells in the presence of APC-like tumours and BMDCs with or without iVAC treatment. Representative flow cytometry plots (a) and quantification (b) of CD8+ T cell proliferation (measured by cell tracer dilution) and activation (CD25+) after 48 h of co-culture with APC-like tumours and BMDCs. c,d, APC-like tumour cells promote the expansion of OT-I naive T cells. Representative flow cytometry plots (c) and quantification (d) of CD8+ T cell proliferation (measured by cell tracer dilution) and activation (CD44+) after 48 h of co-culture with APC-like tumours. e, Quantification of early activation markers CD69+CD25+ and CD44+ on CD8+ T cells after 12 h of co-culture with APC-like tumour cells. In these experiments, the MC38 and B16 cells utilized were all murine PD-L1 knockout cells that expressed human PD-L1. For b, c and e, data were derived from three biological replicates and are presented as mean ± s.d. Statistics: one-way ANOVA (b, d, e) with Tukey’s multiple comparisons test. NS: not significant.

Source data

Extended Data Fig. 8 iVAC induces tumours to acquire APC-like functions via PD-L1 degradation and OVA antigen presentation.

a,b, Representative flow cytometry plots (a) and quantification (b) of OVA (SIINFEKL) tetramer+ CD8+ T cells in PBMCs from mice immunized with OVApep (n = 5 mice per group). c, iVAC treatment induced PD-L1 degradation similar to GlueBody2-CPP and resulted in increased infiltration of CD3+ and CD11c+ cells in tumours. Data are representative of three mice per group. Scale bar: 100 μm. d,e, Representative flow cytometry plots (d) and quantification (e) of tumour PD-L1 levels (n = 3 mice per group). f,g, Representative flow cytometry plots (f) and quantification (g) of OVApep-MHC-I complexes on tumour cells (n = 3 mice per group). Data are the mean ± s.d. Statistics: the unpaired two-sided Student’s t-test (b) and one-way ANOVA (e, g) with Tukey’s multiple comparisons test. NS: not significant.

Source data

Extended Data Fig. 9 Evaluation of the responses against virus epitopes in tumour patients.

a–c, The secreted IFN-γ was measured via ELISA after 6–9 days of stimulation with 7 epitopes separately derived from CMV, EBV, or influenza virus antigens. The responses of selected virus-specific T cells in patients with HLA-A*02:01 (a), HLA-A*11:01 (b), and HLA-A*24:02 (c) were ascertained by comparing them (red) with the irrelevant control OVApep (grey). Data are derived from two or three independent biological replicates and are presented as mean ± s.d.

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Extended Data Fig. 10 Evaluation of the iVAC strategy in a PTC model.

a, Evaluation of the iVAC-mediated T cell redirection and activation. The CRC PTCs (patient 1, HLA-A*02:01) were treated with iVAC or controls. TNF-α as well as IFN-γ were measured by ELISA and tumour inhibition was determined by testing the PTC viability after 7 days (n = 3 biological replicates). b, Tumour inhibition on PTCs from other patients (patients 15–17) was evaluated by testing the PTC viability after 7 days (n = 3 biological replicates). c, Percentage of PD-L1+ tumour cells (within EpCAM+ primary tumour cells) as determined by flow cytometry. Patients exhibiting a response to iVAC are highlighted in red. d, Response rates to iVAC treatment in tumours stratified as PD-L1low (<20%) and PD-L1high (≥20%), demonstrating a correlation between PD-L1 expression level and treatment efficacy. Data are the mean ± s.d. Statistics: one-way ANOVA (a) with Tukey’s multiple comparisons test and the unpaired two-sided Student’s t-test (b). NS: not significant.

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Han, Y., Ma, Y., Pei, M. et al. Intratumoural vaccination via checkpoint degradation-coupled antigen presentation. Nature (2026). https://doi.org/10.1038/s41586-025-09903-1

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