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An antibody–toxin conjugate targeting CD47 linked to the bacterial toxin listeriolysin O for cancer immunotherapy

Abstract

Antigen-presenting cells phagocytose tumor cells and subsequently cross-present tumor-derived antigens. However, these processes are impeded by phagocytosis checkpoints and inefficient cytosolic transport of antigenic peptides from phagolysosomes. Here, using a microbial-inspired strategy, we engineered an antibody–toxin conjugate (ATC) that targets the ‘don’t eat me’ signal CD47 linked to the bacterial toxin listeriolysin O from the intracellular bacterium Listeria monocytogenes via a cleavable linker (CD47–LLO). CD47–LLO promotes cancer cell phagocytosis by macrophages followed by LLO release and activation to form pores on phagolysosomal membranes that enhance antigen cross-presentation of tumor-derived peptides and activate cytosolic immune sensors. CD47–LLO treatment in vivo significantly inhibited the growth of both localized and metastatic breast and melanoma tumors and improved animal survival as a monotherapy or in combination with checkpoint blockade. Together, these results demonstrate that designing ATCs to promote immune recognition of tumor cells represents a promising therapeutic strategy for treating multiple cancers.

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Fig. 1: Conjugation and purification of anti-CD47 antibody with LLO.
Fig. 2: CD47–LLO promotes phagocytosis, lysosomal permeabilization and antigen presentation in vitro.
Fig. 3: CD47–LLO activates STING signaling in vitro and in primary breast cancer in vivo.
Fig. 4: CD47–LLO drives tumor antigen-driven T cell responses in vivo.
Fig. 5: Transcriptome analysis reveals CD47–LLO TAM pro-inflammatory signatures.
Fig. 6: CD47–LLO facilitates innate and adaptive cell clustering and signaling in vivo.
Fig. 7: CD47–LLO requires macrophages and CD8+ T cells for tumor cell elimination.
Fig. 8: CD47–LLO drives systemic anti-tumor immunity to inhibit breast cancer metastasis.

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Data availability

Data supporting the findings of this study are available within the article and its Supplementary Information. The sequencing data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus database under the publicly available accession number GSE255937. The GRCm39-based mouse reference genome is available from the UCSC Genome Browser (http://genome.ucsc.edu). Source data are provided with this paper.

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Acknowledgements

We thank K. Duprez and F. Han (Structure Based Design, Inc.) and Bio-Synthesis, Inc. for expertise with protein–antibody conjugation, K. Dunner, Jr (High Resolution Electron Microscopy Facility) for help with transmission electron microscopy image acquisition, D. Fisher (Massachusetts General Hospital) for the gift of the D4M.3A melanoma cell line, J. Zhang of MD Anderson’s Department of Experimental Radiation Oncology for processing histologic samples; V. Van and K. L. Maldonado at MD Anderson’s Small Animal Imaging Facility for helping with the animal experiments, N. R. Vaughn and N. Nguyen at MD Anderson’s Flow Cytometry and Cellular Imaging Core Facility for helping with flow cytometry experiments, C. Shi and J. Yan at MD Anderson’s Oncology Research for Biologics and Immunotherapy Translation platform for helping with ADA experiments and C. Wogan from MD Anderson’s Division of Radiation Oncology for editorial help. This work was supported in part by the National Institutes of Health (R01NS117828 to W.J.) and the American Cancer Society (RSG-22-052-01-IBCD to W.J.), the Radiological Society of North America Resident Grant (to B.R.S.), the SITC Merck Cancer Immunotherapy Clinical Fellowship (to B.R.S.), the American Society of Clinical Oncology Young Investigator Award (to B.R.S.), the American Cancer Society award (PF-24-1156745-01-ET, grant https://doi.org/10.53354/ACS.PF-24-1156745-01-ET.pc.gr.193703 (to B.R.S.)), the Susan G. Komen Foundation Career Transition Award (to Y.W.) and the ABTA and Uncle Kory Foundation Fellowship (to K.H.).

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Authors and Affiliations

Authors

Contributions

W.J., B.Y.S.K., B.R.S. and Y.W. conceived the project and were responsible for all phases of the research. B.R.S. conducted the majority of the experiments and data analyses. Y.W., A.W., N.T., D.L., J.E., K.H., S.D., J.H., Y.M., A.G., S.D.J., M.C., M.K., T.D.G., A.C.K., J.L. and A.A. assisted with data collection and interpretation. N.T. and K.Y. performed bioinformatics analysis. The manuscript was drafted by B.R.S., B.Y.S.K. and W.J. and was revised and approved by all authors.

Corresponding authors

Correspondence to Betty Y. S. Kim or Wen Jiang.

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A provisional patent application based on the technology described in the paper has been filed by the Board of Regents, the University of Texas System, with W.J., B.R.S., Y.W. and B.Y.S.K. as inventors. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Conjugation of anti-CD47 and IgG1 to Listeriolysin O.

a, Results of sheep red blood cell (sRBC) haemolysis assay of purified LLO. Data show n = 3 biologically independent experiments. b, The MIAP410 antibody (BioXCell, Cat#BE0283) or the IgG1 antibody (BioXCell, Cat#BE0083) was dialyzed against phosphate-buffered saline (PBS) buffer, pH 7.2, and the dialyzed antibody was then modified with the click chemistry labelling reagent DBCO-PEG4-NHS ester (Conju-Probe, SKU# CP-2028) for 30 min at room temperature. The crosslinker in excess was removed by dialysis against PBS buffer, pH 7.2. c, The buffer of Listeriolysin O protein (Genscript) was exchanged with PBS buffer, pH 7.2, by PD-10 column (GE healthcare, Cat# 17085101), followed by modification with the click chemistry crosslinking reagent SPDP-PEG11-azide (BroadPharm, Cat# BP-25143). d, SDS-PAGE results of IgG-LLO conjugate in reductive and non-reductive loading buffers. Representative gel of n = 4 biological replicates shown.

Source data

Extended Data Fig. 2 In vitro toxicity assays of CD47-LLO with cancer cell lines.

a-h, Flow cytometry analysis and quantification of apoptosis and necrosis in 4T1Br4 (a-b), EO771 (c-d), KPC (e-f), and D4M.3 A (g-h) cells after treatment with 2 μg/mL CD47-LLO for 0, 6, or 24 hours (n = 4 biologically independent experiments for b, d, f, and n = 3 for h). Data shown represent mean ± s.d. (b, d, f, h) analyzed by one-way analysis of variance with Tukey’s multiple comparisons test.

Source data

Extended Data Fig. 3 CD47-LLO promotes dendritic cell phagocytosis, lysosomal permeabilization, antigen presentation, and cGAS-STING activation in vitro.

a, Flow cytometry analysis and b, quantification of phagocytic activity of bone marrow-derived dendritic cells (BMDCs) as evaluated by flow cytometry. BMDCs were collected from n = 3 C57BL6 mice. c, Flow cytometry analysis and d, quantification of BMDCs stained with acridine orange. BMDCs were collected from n = 4 C57BL6 mice for IgG treatment and n = 3 for remaining treatment groups.  e, Flow cytometry analysis and f, quantification of cross-presentation of SIINFEKL–H2Kb peptides on the surfaces of BMDCs (n = 3). g, Flow cytometry analysis and h, quantification of pSTING levels in BMDCs isolated from co-cultures with EO771 cells. n = 4 C57BL6 mice. Data shown represent mean ± s.d. (b, d, f, h) analyzed by one-way analysis of variance with Tukey’s multiple comparisons test.

Source data

Extended Data Fig. 4 Antitumour effect of intratumoural CD47-LLO in 4T1Br4 and EO771 models.

a, Schematic of experiments involving intratumoural (IT) injections of CD47-LLO or anti-CD47 in syngeneic orthotopic 4T1Br4 breast cancer models. b, 4T1Br4 tumour volumes after IT injection of anti-CD47 or CD47-LLO. n = 4 for intratumoural CD47-LLO, n = 4 for intratumoural anti-CD47. c-d, Flow cytometry analysis of CD4+ (c) and CD8+ (d) tumour-associated lymphocytes in EO771 tumours at day 16 after tumour inoculation in each group. e, Flow cytometry analysis of SIINFEKL–H2Kb tetramer+CD8+ T cells within the tumour microenvironment. Data shown represent mean ± s.e.m. (b) analyzed by two-way analysis of variance with Tukey’s multiple comparisons test. Panel a created by modifying graphics from BioRender.com.

Source data

Extended Data Fig. 5 Biodistribution of CD47-LLO after intratumoural and intraperitoneal administration in vivo.

a, Representative fluorescence images and b, quantification of EO771 tumour-bearing mice taken at predetermined times after intratumoural injection of IR800CW-tagged CD47-LLO (25 μg). n = 2 mice. Bilateral tumours enclosed in circles. c, Ex vivo fluorescence images and d, quantification of tumour and major organs collected at 24 h after intratumoural administration. n = 2 mice. e, Quantification of EO771 tumours taken at predetermined times after intraperitoneal injection of IR800CW-tagged CD47-LLO (100 μg). n = 4 mice. f, Ex vivo fluorescence images and g, quantification of tumour and major organs collected at 36 h after intraperitoneal administration. n = 4 mice. Data shown represent mean ± s.d. (b, d, e, g) analyzed by two-sided unpaired Student’s t test (g).

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Extended Data Fig. 6 Tumour-associated CD45+ cells detected by single-cell RNA sequencing.

a, Uniform manifold approximation and projection (UMAP) of all single cells from 3 treatment groups color-coded by cluster from IgG (n = 3), anti-CD47 (n = 4), or CD47-LLO (n = 4) treated tumours. b, Heatmap of 20 differentially expressed genes in clusters, ranked by false discovery rate (FDR) from the 3 treatment groups. c, Dot plot showing marker expression for different clusters from the 3 treatment groups. Dot size indicates the percentage of cells in each cluster expressing the gene and colors indicate the average expression levels.

Extended Data Fig. 7 Tumour-associated neutrophils are enriched in CD47-LLO tumours.

a, Representative images show levels of Ly-6g+ (top) and Ly-6g + /Cd11b+ (bottom) cells detected by immunostaining in 4T1Br4 breast tumour frozen tissue sections. Scale bar, 10 μm. b, Quantification of Ly-6g+ cells per DAPI+ cells per field of view (n = 15 per treatment condition from n = 3 biologically independent tumours per condition). c, Uniform manifold approximation and projection (UMAP) of only CD45+ granulocytes color-coded by sample from IgG (n = 3), anti-CD47 (n = 4), or CD47-LLO (n = 4) treated tumours. d, UMAP projection of only CD45+ granulocytes color-coded by cluster. e, Numbers of cells (y-axis) from each cluster (x-axis) color-coded by sample. f, Percentage of cells (y-axis) from each cluster (x-axis) color-coded by sample. g, Dot plot depicting the top 5 differentially expressed genes per granulocyte cluster. The dot size indicates the percentage of cells in each cluster expressing the gene and colors indicate the average expression levels. Data shown represent mean ± s.d. (b) analyzed by two-sided unpaired Student’s t test.

Source data

Extended Data Fig. 8 Differentially expressed genes define tumour-associated macrophages clusters.

a, Dot plot depicting the top 10 differentially expressed genes per macrophage cluster. Dot size indicates the percentage of cells in each cluster expressing the gene and colors indicate the average expression levels from IgG (n = 3), anti-CD47 (n = 4), or CD47-LLO (n = 4) treated tumours. b, Dot plot depicting the differential expression of relevant genes for cluster assignments per macrophage cluster from the 3 treatment groups. c, Gene set enrichment analysis utilizing the Gene Ontology Biological Process (GOBP) gene set for tumour-associated macrophages with heatmap displaying the ten most upregulated and downregulated pathways in each cluster ranked by their normalized enrichment scores (NES) from the three treatment groups.

Extended Data Fig. 9 Tumour-infiltrating CD8 T cells show decreased PD-1 positivity with CD47-LLO and anti-PD-1 treatment.

a, Schema for generating in vivo syngeneic orthotopic breast cancer models for intraperitoneal injection of CD47-LLO, anti-CD47, and anti-PD1. b, Flow cytometry analysis and c, quantification of PD-1 negativity in CD8 T cells isolated from 4T1Br4 tumours treated with anti-CD47, CD47-LLO, anti-CD47 + anti-PD-1, or CD47-LLO + anti-PD-1. Data show mean ± s.d. (c) analyzed by one-way analysis of variance with Tukey’s multiple comparisons test. Data show n = 4 mice per treatment group.

Source data

Extended Data Fig. 10 In vivo toxicity of CD47-LLO.

a-b, Lymphocyte (a) and red blood cell (b) counts at day 2 and day 9 after intraperitoneal injection of drug (50 μg IgG, 50 μg anti-CD47, 100 μg CD47-LLO, or 100 μg CD47-LLO + 200 μg anti-PD1). Data show n = 4 mice for IgG and anti-CD47 treatment groups and n = 3 for other treatment groups. c, Serum blood urea nitrogen (BUN) levels and d, serum aspartate transaminase / alanine transaminase (AST/ALT) levels at day 9 after drug injection. Data show n = 4 mice for IgG and anti-CD47 treatment groups and n = 3 for other treatment groups. e, Body weight changes in mice at day 2 and day 9 after drug injection. Data show n = 4 mice for IgG and anti-CD47 treatment groups and n = 3 for other treatment groups. f, Hematoxylin and eosin staining of paraffin sections of major organs two days after intraperitoneal injection of CD47-LLO or CD47-LLO and anti-PD-1. Experiment was repeated independently n = 3 times with similar results; a representative result is shown. Scale bar, 200 mm. g, Serum IL-6 levels at 4 hours after drug injection measured by enzyme-linked immunosorbent assay (ELISA). Data show n = 4 C57BL6 mice per treatment group. h, Serum IL-1β levels at 4 hours after drug injection measured by ELISA. Data show n = 4 C57BL6 mice per treatment group. i, Serum concentrations of anti-LLO antibody at 6 weeks after drug injection by sandwich immunogenicity assay. Data show n = 3 mice per IgG treatment group and n = 5 mice per CD47-LLO treatment group. Data shown represent mean ± s.d. (a, b, c, d, e, g, h, i) analyzed by one-way analysis of variance with Tukey’s multiple comparisons test (a, b, c, d, e) or two-sided unpaired Student’s t test (g, h, i).

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Schrank, B.R., Wang, Y., Wu, A. et al. An antibody–toxin conjugate targeting CD47 linked to the bacterial toxin listeriolysin O for cancer immunotherapy. Nat Cancer 6, 511–527 (2025). https://doi.org/10.1038/s43018-025-00919-0

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  • DOI: https://doi.org/10.1038/s43018-025-00919-0

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