A logic-gated trispecific engager enhances macrophage killing of cancer cells in solid tumors

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A logic-gated trispecific engager enhances macrophage killing of cancer cells in solid tumors

Data availability

Source data used to generate manuscript figures are provided with this paper or available from a public data repository. RNA-seq generated in this study were deposited to the Genome Sequence Archive database under the accession codes CRA037763 and CRA037764. Source data are provided with this paper.

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Acknowledgements

We acknowledge the technical support from the Advanced Medical Research Institute/Translational Medicine Core Facility of the Advanced Medical Research Institute, Shandong University. We are also grateful for the technical support from the Pharmaceutical Biology Sharing Platform, School of Pharmaceutical Sciences, Shandong University. This work was supported by the National Natural Science Foundation of China grants 82425056 (X.J.), 82350125 (X.J.), 22101154 (K.Z.) and 82173763 (X.J.), the National Key Research and Development Program of China grant 2024YFA0918400 (X.J.), the Fundamental Research Funds of Shandong Province grant ZR2022ZD18 (X.J.), the Shandong Province Excellent Youth Science Fund Project (overseas) grant 2022HWYQ-008 (K.Z.), the Youth Fund from Natural Science Foundation of Shandong Province grant ZR2021QB038 (K.Z.), the Taishan Scholar Program of Shandong Province grants tsqnz20221166 (W.J.) and tsqnz20221165 (Y.Z.), the Natural Science Foundation of Shandong Province grant ZR2023QH001 (W.J.) and the National Natural Science Foundation of China grant 82303810 (Y.Z.). Several illustrations for this manuscript were created with BioRender.com.

Author information

Author notes

  1. These authors contributed equally: Xiaotian Zhao, Weiqiang Jing, Ganyu Wang.

Authors and Affiliations

  1. 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), School of Pharmaceutical Sciences; Department of Urology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China

    Xiaotian Zhao, Weiqiang Jing, Ganyu Wang, Zhanyan Liu, Xinxin Xu, Maosen Han, Zhipeng Fu, Zuolin Zheng, Jing Zhang, Longyu Bo, Xianghui Dong, Caiping Li, Fabao Zhao, Kun Zhao & Xinyi Jiang

  2. Department of Neurosurgery, Qilu Hospital and Institute of Brain and Brain-Inspired Science, Cheeloo College of Medicine, Shandong University, Jinan, China

    Yulin Zhang

  3. Shandong Provincial Key Laboratory of Microparticles Drug Delivery Technology, Qilu Pharmaceutical Co., Ltd., Jinan, China

    Yanhua Sun

  4. State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, China

    Junfeng Zhang

  5. Department of Urology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Nianzeng Xing

Authors

  1. Xiaotian Zhao
  2. Weiqiang Jing
  3. Ganyu Wang
  4. Zhanyan Liu
  5. Xinxin Xu
  6. Maosen Han
  7. Zhipeng Fu
  8. Yulin Zhang
  9. Zuolin Zheng
  10. Jing Zhang
  11. Longyu Bo
  12. Xianghui Dong
  13. Caiping Li
  14. Yanhua Sun
  15. Junfeng Zhang
  16. Fabao Zhao
  17. Nianzeng Xing
  18. Kun Zhao
  19. Xinyi Jiang

Contributions

X.Z., W.J., G.W. and X.X. conceptualized the study and designed experiments. X.Z., Z.L., W.J. and G.W. performed the animal experiments. Z.F, M.H., Y.Z., Z.Z., J.Z. L.B., F.Z, Y.S., X.D., N.X. and C.L. contributed to data analysis and interpretation. X.Z., W.J. and G.W. crafted all the figures and wrote the manuscript. X.J., K.Z., W.J., J.F.Z., N.X. and X.Z. edited and revised the manuscript and supervised the research. The final draft of the manuscript was approved by all coauthors.

Corresponding authors

Correspondence to Weiqiang Jing, Junfeng Zhang, Fabao Zhao, Nianzeng Xing, Kun Zhao or Xinyi Jiang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Biotechnology thanks Ming Li, Jiaxin Li, Peng Wu 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

Extended Data Fig. 1 Computational-guided screening of cis-targeting αSIRPα-CRT constructs.

a, Schematic of macrophage-mediated clearance, guided by CRT. Created in BioRender. A, H. (2026) https://BioRender.com/k650onf. b, Scatter plot showing macrophage phagocytosis versus the ratio of the mean fluorescence intensity of CRT and CD47 (MFICRT/MFICD47). The trend line represents the linear fit between the phagocytosis and the MFICRT/MFICD47 ratio. Correlation analysis was performed using Pearson’s correlation coefficient. The correlation coefficient (r) and corresponding two-sided P value are shown in the plot (r = 0.8521, P = 0.0001). c, Schematic of the side effects of trans-targeting. Cell cross-linking (left); Lack of synergy (right). Created in BioRender. A, H. (2026) https://BioRender.com/k650onf. d, Representative images of αSIRPα-CRT-SIRPα complexes from the first-tier screening, with FFFF and FFFFRRR shown as representative constructs excluded during this tier. e, Representative images of αSIRPα-CRT constructs from the second-tier screening, with RRR shown as a representative construct excluded during this tier.

Source data

Extended Data Fig. 2 Validation of the superior activity of the cis-targeting αSIRPα-CRT construct.

a, Flow cytometric analysis of the binding efficiency of αSIRPα-CRT constructs to macrophages preincubated with anti-SIRPα scFv, as determined using a Cy5 label. b, Representative flow cytometry and quantitative analysis of the binding efficiency of αSIRPα-FITC to macrophages preincubated with anti-SIRPα scFv. P values, one-way ANOVA. Error bars, mean ± s.d. n = 6 cell samples per group, biological replicates. c, d, Representative flow cytometry (c) and quantitative analysis (d) of the MFILRP1-act in SIRPα-knockout macrophages, which were co-cultured with LRP1-knockout macrophages, under treatment with αSIRPα-CRT (0.05 μg ml−1) for 0.5 h. P values, one-way ANOVA. Error bars, mean ± s.d. n = 6 cell samples per group, biological replicates. Box plots show the median (center line), the 25th and 75th percentiles (box bounds), and whiskers indicate the minimum and maximum values. Individual data points are shown. e, Quantitative analysis of mean fluorescence intensity of activated LRP1 (MFILRP1-act) in macrophages treated with different αSIRPα-CRT constructs (0.05 μg mL−1) for 0.5 h. P values, one-way ANOVA. n = 6 cell samples per group, biological replicates. f, Quantitative analysis of mean fluorescence intensity of activated SIRPα (MFISIRPα-act) in macrophages treated with different αSIRPα-CRT constructs (0.05 μg mL−1) for 0.5 h. P values, one-way ANOVA. n = 6 cell samples per group, biological replicates. g, Quantitative analysis of the ratio of MFILRP1-act to MFISIRPα-act. P values, one-way ANOVA. Error bars, mean ± s.d. n = 6 cell samples per group, biological replicates. h, i, Representative flow cytometry (h) and quantitative analysis (i) of BMDM phagocytosis of CD47-conjugated fluorescent beads with αSIRPα-CRT treatment. P values, one-way ANOVA. Error bars, mean ± s.d. n = 3 cell samples per group, biological replicates. j, The ratio of mean fluorescence intensity of activated LRP1 to activated SIRPα (MFILRP1-act/MFISIRPα-act) in macrophages treated with αSIRPα-CRT (FRF) or CRT-αSIRPα (FRF) for 0.5 h. P values, one-way ANOVA. Error bars, mean ± s.d. n = 5 cell samples per group, biological replicates. k, Confocal images of macrophages cultured in serum-free medium on ultra-low attachment plates after 12 h of treatment with αSIRPα-CRT (FRF) or CRT-αSIRPα (FRF). n = 3 cell samples per group, biological replicates. l, m, Representative flow cytometry (l) and quantitative analysis (m) of BMDM phagocytosis of CD47-conjugated fluorescent beads with CRTtrun-αSIRPα treatment. P values, one-way ANOVA. Error bars, mean ± s.d. n = 4 cell samples per group, biological replicates. αSIRPα-CRT (Anti-SIRPα–CRT), αSIRPα-FITC (Anti-SIRPα–FITC), CRT-αSIRPα (CRT–anti-SIRPα). ns, not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are indicated in the graph.

Source data

Extended Data Fig. 3 Evaluation of the in vitro effects of TrME on macrophage phagocytosis and antigen presentation.

a, Quantitative analysis of the ratio of the mean fluorescence intensity of activated LRP1 to activated SIRPα (MFILRP1-act/MFISIRPα-act) in macrophages treated with different TrMEB7H3 constructs (0.05 μg mL−1) for 0.5 h. P values, one-way ANOVA. Error bars, mean ± s.d. n = 6 cell samples per group, biological replicates. b, c, TrMEB7H3 and RRR-linked TrMEB7H3-mediated phagocytosis of GL261 cells (b) and mouse red blood cells (RBC) (c) by macrophages, in the presence or absence of CD47-conjugated beads. P values, one-way ANOVA. Error bars, mean ± s.d. n = 4 cell samples per group, biological replicates. Box plots show the median (center line), the 25th and 75th percentiles (box bounds), and whiskers indicate the minimum and maximum values. Individual data points are shown. d, Confocal images of macrophages cultured in serum-free medium on ultra-low attachment plates after 12 hours of various treatment. n = 3 cell samples per group, biological replicates. e, Flow cytometry analysis and quantification of CD70-expressing GL261 cell phagocytosis by BMDMs (after 4 h of incubation) in the presence of TrMECD70 (0.1 μg ml−1). P values, one-way ANOVA. Error bars, mean ± s.d. n = 5 cell samples per group, biological replicates. f, Confocal images of antigen presentation levels of H2kb-SIINFEKL complexes in BMDMs which were cocultured with GL261-cOVA cells or GL261B7H3–cOVA cell, under treatment with TrMEB7H3. Green, BMDMs; Red, H2kb-SIINFEKL complex. n = 4 cell samples per group, biological replicates. g, Elispot assay for the detection of IFN-γ+ cells in OT-Ⅰ T cells co-cultured with GL261-cOVA-TrMEB7H3-BMDMs or GL261B7H3–cOVA-TrMEB7H3-BMDMs systems. h, Quantitative bar graphs of IFN-γ+ cell spots in (g). P values, one-way ANOVA. Error bars, mean ± s.d. n = 3 cell samples per group, biological replicates. ns, not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are indicated in the graph.

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Extended Data Fig. 4 The phenotypic polarization of macrophages after TrME treatment.

a, Heatmap of M1 and M2-related biomarkers of sorted BMDMs from GL261-TrMEB7H3-BMDMs coculture system. n = 3 cell samples per group, biological replicates. b, Heatmap of M1 and M2-related biomarkers of sorted BMDMs from TrMEB7H3-BMDMs or TrMEB7H3-BMDMs-GL261 coculture systems. n = 3 cell samples per group, biological replicates. c-g, Quantitative bar graphs of MHC Ⅱ (H2-Ab1) (c), CLEC-1 (d), PD-L2 (e), 4-1BBL (f), and OX40L (g) gene expression of sorted BMDMs from GL261-TrMEB7H3-BMDMs coculture system via RNA-seq analysis. P values, two-sided unpaired t-test. Error bars, mean ± s.d. n = 3 cell samples per group, biological replicates. h, GO pathway analysis of up- and down-regulated pathways in sorted BMDMs from GL261-TrMEB7H3-BMDMs coculture system. GO pathway enrichment analysis of differentially expressed genes was performed using the clusterProfiler package. Enrichment significance was assessed using a hypergeometric test, and P values were adjusted for multiple comparisons using the Benjamini–Hochberg false discovery rate (FDR) method. GO terms with Padj < 0.05 were considered significantly enriched. ns, not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are indicated in the graph.

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Extended Data Fig. 5 Efficacy evaluation of single-dose TrME formulations in an orthotopic GBM mouse model.

a, Experimental schedule for in vivo studies with single dose of 2.5 mg kg−1 TrMEB7H3-protein. Created in BioRender. A, H. (2026) https://BioRender.com/k650onf. b, Representative IVIS imaging of GBM-bearing mice following the designated treatments on days 7, 12, 17, and 22. Representative pictures from n = 6 mice per group, biological replicates. c, Quantification of the bioluminescence signal intensity of GBM-bearing mice following the designated treatments on days 7, 12, 17, and 22. P values, two-way ANOVA. Error bars, mean ± s.d. n = 6 mice per group, biological replicates. d, Survival of GL261 tumor-bearing mice treated with TrMEB7H3-protein or PBS control. Survival was estimated by the Kaplan-Meier method and compared by log-rank test. n = 6 mice per group, biological replicates. e, Experimental schedule for in vivo studies with single dose of TrMEB7H3-MC3. Created in BioRender. A, H. (2026) https://BioRender.com/k650onf. f, Representative IVIS imaging of GBM-bearing mice following the designated treatments on days 7, 12, 17, and 22. Representative pictures from n = 6 mice per group, biological replicates. g, Quantification of the bioluminescence signal intensity of GBM-bearing mice following the designated treatments on days 7, 12, 17, and 22. P values, two-way ANOVA. Error bars, mean ± s.d. n = 6 mice per group, biological replicates. h, Survival of GL261 tumor-bearing mice treated with TrMEB7H3-MC3, TrMEB7H3-protein or MC3 control. Survival was estimated by the Kaplan-Meier method and compared by log-rank test. n = 6 mice per group, biological replicates. ns, not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are indicated in the graph.

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Extended Data Fig. 6 Screening of Factors Determining LNP Delivery Efficiency.

a, GL261 cells were treated with firefly luciferase mRNA (mLuc)-loaded LNPs based on BC-9 and BT-9. The luminescence intensity of luciferase after 24 h incubation with mLuc-LNPs was shown in the heat map. Compositions A, B, C, and D were detailed in Supplementary Fig. 9b. Each set of 16 numbers corresponded to the lipid molar ratios listed in Supplementary Fig. 9c. b, Statistical analysis of the delivery efficiency of BT-9 LNPs and BC-9 LNPs. Data sourced from Extended Data Fig. 6a. Compositions A, B, C, and D were detailed in Supplementary Fig. 9b. P values, two-sided paired t-test. n = 16 cell samples, biological replicates with identical LNP compositions but different lipid molar ratios. c, Statistical analysis of the delivery efficiency of DOPE LNPs and DPPC LNPs after grouping based on ionizable lipids and PEG lipids. Data sourced from Extended Data Fig. 6a. P values, two-sided paired t-test. n = 16 cell samples, biological replicates with identical LNP compositions but different lipid molar ratios. d, Statistical analysis of the delivery efficiency of C14PEG2k LNPs and DMG-PEG2k LNPs after grouping based on ionizable lipids and helper lipids. Data sourced from Extended Data Fig. 6a. P values, two-sided paired t-test. n = 16 cell samples, biological replicates with identical LNP compositions but different lipid molar ratios. e, Statistical analysis of the delivery efficiency of BC-9 LNPs, BC-12 LNPs, BC-14 LNPs, and BC-16 LNPs. Data sourced from Fig. 3e. Compositions A, E, F, and D were detailed in Fig. 3d. n = 16 cell samples, biological replicates with identical LNP compositions but different lipid molar ratios. f, Statistical analysis of the delivery efficiency of C14PEG2k LNPs and C18PEG2k LNPs after grouping based on ionizable lipids and helper lipids. Data sourced from Fig. 3e. P values, two-sided paired t-test. n = 16 cell samples, biological replicates with identical LNP compositions but different lipid molar ratios. g, Statistical analysis of the delivery efficiency of DOPE LNPs and DOTAP LNPs after grouping based on ionizable lipids and PEG lipids. Data sourced from Fig. 3e. P values, two-sided paired t-test. n = 16 cell samples, biological replicates with identical LNP compositions but different lipid molar ratios. ns, not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are indicated in the graph.

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Extended Data Fig. 7 Evaluation of hematological toxicity of single-dose and multiple-dose TrME formulations.

a-d, Complete blood counts were performed three days after mice received the single-dose treatments (2.5 mg kg−1 TrMEB7H3-protein, αSIRPα-CRT (Anti-SIRPα–CRT) or anti-CD47 antibody; 0.5 mg kg−1 TrMEB7H3-BCT-A5). n = 10 mice per group, biological replicates. a, Red blood cells (RBCs). P values, one-way ANOVA. Error bars, mean ± s.d. b, Hemoglobin (HGB). P values, one-way ANOVA. Error bars, mean ± s.d. c, Platelet (PLTs). P values, one-way ANOVA. Error bars, mean ± s.d. d, White blood cells (WBCs), Neutrophils (NEUs), Lymphocytes (LYMs), Monocytes (MONOs). P values, two-way ANOVA. Error bars, mean ± s.d. e-h, Mice received either a single-dose treatment or a repeated dosing regimen consisting of injections every five days for a total of three times. (2.5 mg kg−1 TrMEB7H3-protein or anti-CD47 antibody). Complete blood counts were performed three days after the single injection or three days after the final dose in the repeated dosing schedule. n = 10 mice per group, biological replicates. e, Red blood cells (RBCs). P values, one-way ANOVA. Error bars, mean ± s.d. f, Hemoglobin (HGB). P values, one-way ANOVA. Error bars, mean ± s.d. g, Platelet (PLTs). P values, one-way ANOVA. Error bars, mean ± s.d. h, White blood cells (WBCs), Neutrophils (NEUs), Lymphocytes (LYMs), Monocytes (MONOs). P values, two-way ANOVA. Error bars, mean ± s.d. ns, not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are indicated in the graph.

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Extended Data Fig. 8 Immunological analysis of the tumor microenvironment in orthotopic GBM mouse models after TrME treatment.

a, Magnetic resonance imaging (MRI) of the brains of GBM-bearing mice after the designated treatments on day 22. The magenta dotted circle points to the tumor site. Representative pictures from n = 6 mice per group, biological replicates. b, H&E staining of brain tissue sections from GBM-bearing mice after the designated treatments on day 22. Representative pictures from n = 6 mice per group, biological replicates. c, Survival of GL261 tumor-bearing mice treated with continued dosing TrMEB7H3-BCT-A5, TrMEB7H3-protein, or saline control. Survival was estimated by the Kaplan-Meier method and compared by log-rank test. n = 10 mice per group, biological replicates. d, TrMEB7H3 concentration in tumors was measured on indicated time points after single injection of TrMEB7H3-BCT-A5 into GL261 GBM mice. P values, one-way ANOVA. Error bars, mean ± s.d. n = 6 mice per group, biological replicates. e, TUNEL staining of brain sections from GBM-bearing mice on day 22 following the designated treatments. Representative pictures from n = 6 mice per group, biological replicates. f, Quantification of CD69+, Granzyme B+, and IFN-γ+ cells in tumor-infiltrating CD8+ T cells. P values, two-way ANOVA. Error bars, mean ± s.d. n = 6 mice per group, biological replicates. g, Flow cytometry data showing the numbers of tumor-infiltrating macrophages, CD8+ T cells, and Tregs, respectively. All the populations are denoted as cell numbers per gram of tumor. P values, two-way ANOVA. Error bars, mean ± s.d. n = 6 mice per group, biological replicates. h, Flow cytometry data showing the numbers of tumor-infiltrating CD86+ CD11b+ F4/80+ cells and CD206+ CD11b+ F4/80+ cells, respectively. All the populations are denoted as cell numbers per gram of tumor. P values, two-way ANOVA. Error bars, mean ± s.d. n = 6 mice per group, biological replicates. i, Flow cytometry data showing the numbers of tumor-infiltrating CD44+ CD8+ T cells, CD69+ CD8+ T cells, Granzyme B+ CD8+ T cells, and IFN-γ+ CD8+ T cells, respectively. All the populations are denoted as cell numbers per gram of tumor. P values, two-way ANOVA. Error bars, mean ± s.d. n = 6 mice per group, biological replicates. j, Flow cytometry data showing the numbers of tumor-infiltrating monocyte (M-)MDSCs (CD11b+ Ly6G− Ly6Chi) and polymorphonuclear (PMN-)MDSCs (CD11b+ Ly6G+ Ly6Clo), respectively. All the populations are denoted as cell numbers per gram of tumor. P values, two-way ANOVA. Error bars, mean ± s.d. n = 6 mice per group, biological replicates. αSIRPα–αB7H3 (Anti-SIRPα–anti-B7H3), CRT–αB7H3 (CRT–anti-B7H3), αSIRPα–CRT (Anti-SIRPα–CRT), αSIRPα/B7H3 (Anti-SIRPα/B7H3). ns, not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are indicated in the graph.

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Extended Data Fig. 9 Evaluation of the therapeutic efficacy of TrME formulations in an orthotopic MB49 bladder cancer mouse model.

a, Photographic images of tumors obtained from mice treated with different therapies. The scale bar represents 1 cm. n = 5 mice per group, biological replicates. b, Statistical graph of the tumor weights. P values, one-way ANOVA. Error bars, mean ± s.d. n = 5 mice per group, biological replicates. c, Photographic images of tumors obtained from mice treated with different therapies. The scale bar represents 1 cm. n = 5 mice per group, biological replicates. d, Statistical graph of the tumor weights. P values, one-way ANOVA. Error bars, mean ± s.d. n = 5 mice per group, biological replicates. e, Photographic images of tumors obtained from mice treated with different therapies. The scale bar represents 1 cm. n = 5 mice per group, biological replicates. f, Statistical graph of the tumor weights. P values, one-way ANOVA. Error bars, mean ± s.d. n = 5 mice per group, biological replicates. CRT–αB7H3 (CRT–anti-B7H3), αSIRPα/B7H3 (Anti-SIRPα/B7H3), αCD47 (Anti-CD47), αSIRPα (Anti-SIRPα) ns, not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are indicated in the graph.

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Extended Data Fig. 10 Analysis of epitope spreading induced by TrME in an orthotopic breast cancer mouse model.

a, Photographic images of bilateral breast tumors obtained from mice after TrMEB7H3-BCT-A5 treatment. n = 6 mice per group, biological replicates. b, Statistical graph of the tumor weights. P values, two-way ANOVA. Error bars, mean ± s.d. n = 6 mice per group, biological replicates. ns, not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P values are indicated in the graph.

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Zhao, X., Jing, W., Wang, G. et al. A logic-gated trispecific engager enhances macrophage killing of cancer cells in solid tumors. Nat Biotechnol (2026). https://doi.org/10.1038/s41587-026-03057-9

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