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Antitumour vaccination via the targeted proteolysis of antigens isolated from tumour lysates

Nature Biomedical Engineering volume 9, pages 234–248 (2025)Cite this article

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Abstract

The activation of cytotoxic T cells against tumour cells typically requires the cross-presentation, by antigen-presenting cells (and via major histocompatibility complex class I molecules), of an epitope derived from a tumour antigen. A critical step in antigen processing is the proteolysis of tumour antigens mediated by the ubiquitin–proteasome pathway. Here we describe a tumour vaccine leveraging targeted antigen degradation to augment antigen processing and cross-presentation. Analogous to proteolysis-targeting chimaeras, the vaccine consists of lymph-node-targeting lipid nanoparticles encapsulated with tumour antigens pre-conjugated with ligands that can bind to E3 ubiquitin ligases. In mice with subcutaneous human melanoma or triple-negative breast cancer, or with orthotopic mouse Lewis lung carcinoma or clinically inoperable mouse ovarian cancer, subcutaneously delivered vaccines prepared using tumour lysate proteins elicited antigen-specific adaptive immunity and immunological memory, and inhibited tumour growth, metastasis and recurrence, particularly when combined with immune checkpoint inhibition.

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Fig. 1: TAgD-TVac enhances antigen cross-presentation and induces robust antitumour immune response.
Fig. 2: Preparation of TAgD-TVac and the enhanced cross-presentation in DCs by improving proteasome-mediated antigen degradation.
Fig. 3: TAgD-TVacOVA targets LNs and induces robust antitumour immunity in vivo.
Fig. 4: TAgD-TVac4T1 induces robust antitumour immunity and inhibits lung metastasis in a breast cancer model.
Fig. 5: TAgD-TVacLLC combined with ICB therapy for orthotopic Lewis lung cancer.
Fig. 6: TAgD-TVacID8 combined with ICB therapy for inoperable ovarian cancer.

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Data availability

All data supporting the findings of this study are available within the article and its Supplementary Information. The raw and analysed datasets generated during the study are available for research purposes from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We acknowledge the financial support by the NIH R01 EB027170-01. We thank J. Chen at the Massachusetts Institute of Technology for proofreading the paper and providing valuable comments.

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, Tufts University, Medford, MA, USA

    Yu Zhao, Donghui Song, Zeyu Wang, Zhongfeng Ye, Douglas Wich, Mengting Chen, Jennifer Khirallah, Shuliang Gao & Qiaobing Xu

  2. Key Laboratory of Functional Polymer Materials of Ministry of Education, State Key Laboratory of Medicinal Chemical Biology, Frontiers Science Center for New Organic Matter, College of Chemistry, Nankai University, Tianjin, China

    Qingqing Huang & Yang Liu

  3. Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China

    Fan Huang

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Contributions

Y.Z. and Q.X. devised the project. Y.Z., D.S., Z.W., Q.H., F.H. and M.C. carried out the experimental work and analysed the data. Z.Y., Z.W. and Y.L. participated in discussion of the results. S.G. provided the TEM images. D.W. and J.K. polished the language. Y.Z. and Q.X. wrote the paper.

Corresponding author

Correspondence to Qiaobing Xu.

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Competing interests

Q.X. and Y.Z. are inventors of a pending patent (PCT/US24/46728) related to this work filed by Tufts University. Q.X. is the founder and consultant of Hopewell Therapeutics Inc. The other authors declare no competing interests.

Peer review

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Nature Biomedical Engineering thanks Betty Kim, Kanyi Pu and Jun Wang for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Characterization of adjuvant LNP and its LN-targeting ability.

a) Particle size of adjuvant LNPs. b) Representative IVIS images of fluorescence distribution in BALB/c mice after SC injection of DiR-labelled adjuvant LNPs at the tail base for 6 hours. c) Quantitative analysis of the bioluminescent signals in LNs. d) In vivo fluorescence images of mouse at different time points after injection of DiR-labelled adjuvant LNPs (the carrier of TAgD-TVac). e) The metabolic profile of DiR-labelled adjuvant LNP. f) The mice subcutaneously treated with adjuvant LNP. g) and h) The levels of inflammatory chemokines in the skin samples collected from the injection site. ALC-0315 LNP was used as comparison. Granulocyte-macrophage colony-stimulating factor, GM-CSF; Interleukin 6, IL-6. i) Fluorescence images of DC2.4 cells after incubating with AHPC-GFP-Cre LNP for 6 hours. LysoTracker Red is added to stain the endo–lysosomes. Scale bar, 10 µm. Data are presented as mean ± s.d. from n independent experiments (n = 3). Statistical significance was analysed by one-way ANOVA with Tukey’s multiple comparisons test.

Source data

Extended Data Fig. 2 TAgD-TVacOVA-mediated proteasomal degradation of OVA in BMDCs.

a) HPLC-MS data to verify the encapsulation of AHPC. b) Flow cytometry analysis of the internalization of different formulations by DC2.4 cells through analysing the fluorescence of GFP. c) and d) Quantitative analysis of co-localization of GFP signals (green) with pVHL signals (red). The coefficients are close to 1 if they are highly colocalized. e) The degradation kinetics of GFP-Cre in DC2.4 cells after incubation with AHPC-GFP-Cre LNPs for 3 hours. The cells pre-treated with free AHPC molecules are employed for comparison. GFP-Cre fluorescence is monitored over 24 hours. f) SDS polyacrylamide gel electrophoresis of OVA and AHPC-OVA. g) Fluorescence spectra of the mixture of AHPC-OVA and pVHL after 1-hour incubation. The mixture of OVA, AHPC, and pVHL was used as comparison. pVHL OVA, and pVHL were pre-labelled with FITC and RITC, respectively. h) Western blotting analysis of OVA levels in the cytoplasm in BMDCs after incubating with different LNP formulations for 3 hours. The OVA levels are evaluated after further 24-hour incubation. LNP/protein = 10/1, w/w. Data are presented as mean ± s.d. from n independent experiments (n = 3). Statistical significance was analysed by t test for c, and two-way ANOVA with Tukey’s test for d.

Source data

Extended Data Fig. 3 The pro-inflammatory cytokines and chemokines in the blood in a melanoma mouse model.

a) The levels of pro-inflammatory cytokines (including M-CSF, IFN-γ, and IL-6) in the blood. Macrophage colony-stimulating factor, M-CSF; Interferon-γ, IFN-γ; Interleukin 6, IL-6. b) The levels of pro-inflammatory chemokines (including MCP-1, MIP-1α, and MIP-1β). Monocyte chemoattractant protein-1, MCP-1; Macrophage inflammatory protein-1α, MIP-1α; Macrophage inflammatory protein-1β, MIP-1β. c) and d) Flow cytometry analysis of the population of CD3+CD8+ T cells that bear T cell receptors binding to H2Kb OVA tetramer-SIINFEKL in tumour tissues. e) and f) Flow cytometry analysis of the population of CD3+CD8+ T cells that bear T cell receptors binding to H2Kb OVA tetramer-SIINFEKL in the LNs. Data are presented as mean ± s.d. from n independent experiments (n = 3). Statistical significance was analysed by one-way ANOVA with Tukey’s multiple comparisons test.

Source data

Extended Data Fig. 4 The prophylactic effect of TAgD-TVacOVA in a melanoma mouse model.

a) Schematic illustration of the timeline for vaccination, bleeding (red arrows), and B16F10-OVA tumour inoculation in a C57BL/6 mouse model. B16F10-OVA cells (0.5 × 106 per mouse) are subcutaneously injected at the left flank of C57BL/6 mice. b) and c) OVA-specific IgM and IgG in the blood at different time points after vaccination of the mice with TAgD-TVacOVA. d) Individual tumour growth curves. CR, complete response. Data are presented as mean ± s.d. from n independent experiments (b and c, n = 3; d, n = 6). Statistical significance was analysed by one-way ANOVA with Tukey’s multiple comparisons test.

Source data

Extended Data Fig. 5 Antitumour immunity induced by TAgD-TVac4T1 in a breast cancer mouse model.

a) Flow cytometry analysis of the expression of CD80 and CD86 in DCs (gated on CD11c+ cells) within LNs. b) Flow cytometry analysis of the population of CD8+ T cells (gated on CD45+CD3+ cells) in tumour tissues. c) Flow cytometry analysis of the expression of GzmBhigh in CD8+ T cells (gated on CD45+CD3+ cells) in tumour tissues. d) and e) Flow cytometry analysis of the CD62LlowCD44high and CD62LhighCD44high T cells (gated on CD3+CD8+ cells) in the spleens. f) and g) Flow cytometry analysis of the population of CD8+ T cells (gated on CD45+CD3+ cells) in lung tissues. h) Average tumour growth kinetics. i) Survival of 4T1-Luc tumour-bearing mice. Data are presented as mean ± s.d. from n independent experiments (e and g, n = 3; h and i, n = 5). Statistical significance was analysed by one-way ANOVA with Tukey’s multiple comparisons test for e and g, and two-way ANOVA with Tukey’s test for h and i.

Source data

Extended Data Fig. 6 Antitumour immunity induced by TAgD-TVacLLC in an orthotopic lung cancer mouse model.

a) Flow cytometry analysis of the expression of CD80 and CD86 in DCs (gated on CD11c+ cells) within LNs. b) and c) Flow cytometry analysis of the CD62LlowCD44high and CD62LhighCD44high T cells (gated on CD3+CD8+ cells) in the spleens. d) and e) Flow cytometry analysis of the population of CD8+ T cells (gated on CD45+CD3+ cells) in LLC-Luc tumour tissues. Data are presented as mean ± s.d. from n independent experiments (n = 3). Statistical significance was analyzed by one-way ANOVA with Tukey’s multiple comparisons test.

Source data

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Zhao, Y., Song, D., Wang, Z. et al. Antitumour vaccination via the targeted proteolysis of antigens isolated from tumour lysates. Nat. Biomed. Eng 9, 234–248 (2025). https://doi.org/10.1038/s41551-024-01285-5

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