Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Dual-target CAR-T therapy for ovarian cancer: synergistic targeting of MSLN and B7H3 enhances anti-tumor efficacy and overcomes antigen heterogeneity

Oncogene volume 45, pages 446–458 (2026)Cite this article

Subjects

Abstract

Ovarian cancer (OC) remains a lethal malignancy with limited treatment options owing to antigen heterogeneity and an immunosuppressive tumor microenvironment (TME). Here, we designed a unique chimeric Antigen Receptor T-Cell (CAR-T) construct (B4M3) that integrates an anti-MSLN scFv linked to the CD3ζ activation domain and an anti-B7H3 scFv linked to the 4-1BB co-stimulatory domain. In vitro, B4M3 CAR-T cells exhibited robust cytotoxicity against OC cell lines with enhanced degranulation (CD107a) and efficient tumor cell killing, even at low effector-to-target ratios. In vivo, B4M3 CAR-T cells significantly inhibited tumor growth and prolonged survival and demonstrated superior tumor infiltration and persistence in OC xenograft models. Imaging mass cytometry (IMC) revealed that B4M3 treatment reshaped the TME, increased cytotoxic T lymphocyte (CTL) infiltration, and reduced regulatory T cells (Tregs). Mechanistically, B4M3 therapy upregulated TGF-β, promoting Th17 differentiation and CTL recruitment, thereby enhancing anti-tumor immunity. Our findings demonstrate that B4M3 CAR-T cells effectively address antigen heterogeneity and enhance therapeutic efficacy in OC, thereby offering a promising strategy for solid tumor immunotherapy.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Characterization of B4M3 CAR-T cells.
Fig. 2: B4M3 CAR-T cell therapy promotes tumor regression in an OC mouse model.
Fig. 3: IMC analysis of tumor-infiltrating immune cell differences in different treatment groups.
Fig. 4: B4M3 treatment enhances cytotoxic immune activity against OC cells.
Fig. 5: Imaging mass spectrometry captures major activation pathways and associated protein types in the OC microenvironment.
Fig. 6: IMC analysis of differences in tumor infiltration of T cells of different phenotypes in different treatment groups.

Similar content being viewed by others

Data availability

The authors declare that all data supporting the results of this study are available in the paper and its supplementary information. Source data for the figures in this study are available from the corresponding author upon request.

References

  1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394–424.

    PubMed  Google Scholar 

  2. Torre LA, Trabert B, DeSantis CE, Miller KD, Samimi G, Runowicz CD, et al. Ovarian cancer statistics, 2018. CA Cancer J Clin. 2018;68:284–96.

    PubMed  PubMed Central  Google Scholar 

  3. Bouchkouj N, Kasamon YL, de Claro RA, George B, Lin X, Lee S, et al. FDA approval summary: axicabtagene ciloleucel for relapsed or refractory large b-cell lymphoma. Clin Cancer Res. 2019;25:1702–8.

    Article  PubMed  Google Scholar 

  4. Munshi NC, Anderson LDJ, Shah N, Madduri D, Berdeja J, Lonial S, et al. Idecabtagene vicleucel in relapsed and refractory multiple myeloma. N Engl J Med. 2021;384:705–16.

    Article  PubMed  CAS  Google Scholar 

  5. Wagner J, Wickman E, DeRenzo C, Gottschalk S. CAR t cell therapy for solid tumors: bright future or dark reality?. Mol Ther. 2020;28:2320–39.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Marofi F, Motavalli R, Safonov VA, Thangavelu L, Yumashev AV, Alexander M, et al. CAR t cells in solid tumors: challenges and opportunities. Stem Cell Res Ther. 2021;12:81.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Klampatsa A, Dimou V, Albelda SM. Mesothelin-targeted CAR-t cell therapy for solid tumors. Expert Opin Biol Ther. 2021;21:473–86.

    Article  PubMed  CAS  Google Scholar 

  8. Scales SJ, Gupta N, Pacheco G, Firestein R, French DM, Koeppen H, et al. An antimesothelin-monomethyl auristatin e conjugate with potent antitumor activity in ovarian, pancreatic, and mesothelioma models. Mol Cancer Ther. 2014;13:2630–40.

    Article  PubMed  CAS  Google Scholar 

  9. Winter JM, Tang LH, Klimstra DS, Brennan MF, Brody JR, Rocha FG, et al. A novel survival-based tissue microarray of pancreatic cancer validates MUC1 and mesothelin as biomarkers. PLoS ONE. 2012;7:e40157.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Ho M, Bera TK, Willingham MC, Onda M, Hassan R, FitzGerald D, et al. Mesothelin expression in human lung cancer. Clin Cancer Res. 2007;13:1571–5.

    Article  PubMed  CAS  Google Scholar 

  11. Baba K, Ishigami S, Arigami T, Uenosono Y, Okumura H, Matsumoto M, et al. Mesothelin expression correlates with prolonged patient survival in gastric cancer. J Surg Oncol. 2012;105:195–9.

    Article  PubMed  CAS  Google Scholar 

  12. Obulhasim G, Fujii H, Matsumoto T, Yasen M, Abe M, Matsuoka S, et al. Mesothelin gene expression and promoter methylation/hypomethylation in gynecological tumors. Eur J Gynaecol Oncol. 2010;31:63–71.

    PubMed  CAS  Google Scholar 

  13. Tozbikian G, Brogi E, Kadota K, Catalano J, Akram M, Patil S, et al. Mesothelin expression in triple negative breast carcinomas correlates significantly with basal-like phenotype, distant metastases and decreased survival. PLoS One. 2014;9:e114900.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Zhao B, Li H, Xia Y, Wang Y, Wang Y, Shi Y, et al. Immune checkpoint of b7-h3 in cancer: from immunology to clinical immunotherapy. J Hematol Oncol. 2022;15:153.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Majzner RG, Theruvath JL, Nellan A, Heitzeneder S, Cui Y, Mount CW, et al. CAR t cells targeting b7-h3, a pan-cancer antigen, demonstrate potent preclinical activity against pediatric solid tumors and brain tumors. Clin Cancer Res. 2019;25:2560–74.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Beatty GL, O’Hara MH, Lacey SF, Torigian DA, Nazimuddin F, Chen F, et al. Activity of mesothelin-specific chimeric antigen receptor t cells against pancreatic carcinoma metastases in a phase 1 trial. Gastroenterology. 2018;155:29–32.

    Article  PubMed  CAS  Google Scholar 

  17. Vitanza NA, Wilson AL, Huang W, Seidel K, Brown C, Gustafson JA, et al. Intraventricular b7-h3 CAR t cells for diffuse intrinsic pontine glioma: preliminary first-in-human bioactivity and safety. Cancer Discov. 2023;13:114–31.

    Article  PubMed  CAS  Google Scholar 

  18. Saif WM, Cassier PA, Curigliano G, Daniele G, Hilton JF, Koganemaru S, et al. A phase 1 dose escalation/expansion study of GSK5764227 (GSK’227), a b7-homolog 3 (b7-h3) protein targeted antibody-drug conjugate (ADC), in patients with advanced solid tumors, including gastrointestinal (GI) cancers. J Clin Oncol. 2025;43:TPS84.

    Article  Google Scholar 

  19. Zhang E, Yang P, Gu J, Wu H, Chi X, Liu C, et al. Recombination of a dual-CAR-modified t lymphocyte to accurately eliminate pancreatic malignancy. J Hematol Oncol. 2018;11:102.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Yin Y, Rodriguez JL, Li N, Thokala R, Nasrallah MP, Hu L, et al. Locally secreted BiTEs complement CAR t cells by enhancing killing of antigen heterogeneous solid tumors. Mol Ther. 2022;30:2537–53.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Hegde M, Mukherjee M, Grada Z, Pignata A, Landi D, Navai SA, et al. Tandem CAR t cells targeting HER2 and IL13ralpha2 mitigate tumor antigen escape. J Clin Invest. 2016;126:3036–52.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Choi BD, Yu X, Castano AP, Bouffard AA, Schmidts A, Larson RC, et al. CAR-t cells secreting BiTEs circumvent antigen escape without detectable toxicity. Nat Biotechnol. 2019;37:1049–58.

    Article  PubMed  CAS  Google Scholar 

  23. Lanitis E, Poussin M, Klattenhoff AW, Song D, Sandaltzopoulos R, June CH, et al. Chimeric antigen receptor t cells with dissociated signaling domains exhibit focused antitumor activity with reduced potential for toxicity in vivo. Cancer Immunol Res. 2013;1:43–53.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Ji F, Yu J, Zhu Y, Lin H, Gao K, Rao M, et al. B4m3 CAR-t cell enhance antitumor activity and non-tumor toxicity in ovarian cancer. Int Immunopharmacol. 2025;159:114919.

    Article  PubMed  CAS  Google Scholar 

  25. Ji F, Xu L, Long K, Zhang F, Zhang M, Lu X, et al. Rabies virus glycoprotein 29 (RVG29) promotes CAR-t immunotherapy for glioma. Transl Res. 2023;259:1–12.

    Article  PubMed  CAS  Google Scholar 

  26. Park AK, Fong Y, Kim S, Yang J, Murad JP, Lu J, et al. Effective combination immunotherapy using oncolytic viruses to deliver CAR targets to solid tumors. Sci Transl Med. 2020;12:eaaz1863.

  27. Zhai Y, Du Y, Li G, Yu M, Hu H, Pan C, et al. Trogocytosis of CAR molecule regulates CAR-t cell dysfunction and tumor antigen escape. Signal Transduct Target Ther. 2023;8:457.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Rodriguez-Marquez P, Calleja-Cervantes ME, Serrano G, Oliver-Caldes A, Palacios-Berraquero ML, Martin-Mallo A, et al. CAR density influences antitumoral efficacy of BCMA CAR t cells and correlates with clinical outcome. Sci Adv. 2022;8:eabo0514.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Ruella M, Korell F, Porazzi P, Maus MV. Mechanisms of resistance to chimeric antigen receptor-t cells in haematological malignancies. Nat Rev Drug Discov. 2023;22:976–95.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Hu Y, Hou J, Jiang Z, Lin Q. Mechanisms of resistance to CAR-t cell therapy in multiple myeloma: latest updates from the 2024 ASH annual meeting. Exp Hematol Oncol. 2025;14:45.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Marchal I. FOXO1 enhances CAR t cell fitness and function. Nat Biotechnol. 2024;42:699.

    PubMed  Google Scholar 

  32. Stewart C, Ralyea C, Lockwood S. Ovarian cancer: an integrated review. Semin Oncol Nurs. 2019;35:151–6.

    Article  PubMed  Google Scholar 

  33. Bu L, Yan J, Wang Z, Ruan H, Chen Q, Gunadhi V, et al. Advances in drug delivery for post-surgical cancer treatment. Biomaterials. 2019;219:119182.

    Article  PubMed  CAS  Google Scholar 

  34. Sommer C, Boldajipour B, Kuo TC, Bentley T, Sutton J, Chen A, et al. Preclinical evaluation of allogeneic CAR t cells targeting BCMA for the treatment of multiple myeloma. Mol Ther. 2019;27:1126–38.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Flugel CL, Majzner RG, Krenciute G, Dotti G, Riddell SR, Wagner DL, et al. Overcoming on-target, off-tumour toxicity of CAR t cell therapy for solid tumours. Nat Rev Clin Oncol. 2023;20:49–62.

    Article  PubMed  CAS  Google Scholar 

  36. Wang Y, Buck A, Piel B, Zerefa L, Murugan N, Coherd CD, et al. Affinity fine-tuning anti-CAIX CAR-t cells mitigate on-target off-tumor side effects. Mol Cancer. 2024;23:56.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Majzner RG, Rietberg SP, Sotillo E, Dong R, Vachharajani VT, Labanieh L, et al. Tuning the antigen density requirement for CAR t-cell activity. Cancer Discov. 2020;10:702–23.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Fry TJ, Shah NN, Orentas RJ, Stetler-Stevenson M, Yuan CM, Ramakrishna S, et al. CD22-targeted CAR t cells induce remission in b-ALL that is naive or resistant to CD19-targeted CAR immunotherapy. Nat Med. 2018;24:20–8.

    Article  PubMed  CAS  Google Scholar 

  39. Shi X, Yang J, Deng S, Xu H, Wu D, Zeng Q, et al. TGF-beta signaling in the tumor metabolic microenvironment and targeted therapies. J Hematol Oncol. 2022;15:135.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Batlle E, Massague J. Transforming growth factor-beta signaling in immunity and cancer. Immunity. 2019;50:924–40.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Lan Y, Moustafa M, Knoll M, Xu C, Furkel J, Lazorchak A, et al. Simultaneous targeting of TGF-beta/PD-l1 synergizes with radiotherapy by reprogramming the tumor microenvironment to overcome immune evasion. Cancer Cell. 2021;39:1388–403.

    Article  PubMed  CAS  Google Scholar 

  42. Doan AE, Mueller KP, Chen AY, Rouin GT, Chen Y, Daniel B, et al. FOXO1 is a master regulator of memory programming in CAR t cells. Nature. 2024;629:211–8.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Carlson CM, Endrizzi BT, Wu J, Ding X, Weinreich MA, Walsh ER, et al. Kruppel-like factor 2 regulates thymocyte and t-cell migration. Nature. 2006;442:299–302.

    Article  PubMed  CAS  Google Scholar 

  44. Jung YW, Kim HG, Perry CJ, Kaech SM. CCR7 expression alters memory CD8 t-cell homeostasis by regulating occupancy in IL-7- and IL-15-dependent niches. Proc Natl Acad Sci USA. 2016;113:8278–83.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Dominguez CX, Muller S, Keerthivasan S, Koeppen H, Hung J, Gierke S, et al. Single-cell RNA sequencing reveals stromal evolution into LRRC15(+) myofibroblasts as a determinant of patient response to cancer immunotherapy. Cancer Discov. 2020;10:232–53.

    Article  PubMed  CAS  Google Scholar 

  46. Levstek L, Janzic L, Ihan A, Kopitar AN. Biomarkers for prediction of CAR t therapy outcomes: current and future perspectives. Front Immunol. 2024;15:1378944.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Garcia-Murillas I, Schiavon G, Weigelt B, Ng C, Hrebien S, Cutts RJ, et al. Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer. Sci Transl Med. 2015;7:302ra133.

    Article  PubMed  Google Scholar 

  48. Li S, Lai H, Liu J, Liu Y, Jin L, Li Y, et al. Circulating tumor DNA predicts the response and prognosis in patients with early breast cancer receiving neoadjuvant chemotherapy. JCO Precis Oncol. 2020;4:PO.19.00292.

  49. Hope JL, Stairiker CJ, Bae E, Otero DC, Bradley LM. Striking a balance-cellular and molecular drivers of memory t cell development and responses to chronic stimulation. Front Immunol. 2019;10:1595.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Michelini Hess, Doedens R, Goldrath AL, Hedrick AW. SM. Differentiation of CD8 memory t cells depends on foxo1. J Exp Med. 2013;210:1189–200.

    Article  PubMed Central  CAS  Google Scholar 

  51. Zhang H, Yang Z, Yuan W, Liu J, Luo X, Zhang Q, et al. Sustained AhR activity programs memory fate of early effector CD8(+) t cells. Proc Natl Acad Sci USA. 2024;121:e2317658121.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Agliardi G, Dias J, Rampotas A, Garcia J, Roddie C. Accelerating and optimising CAR t-cell manufacture to deliver better patient products. Lancet Haematol. 2025;12:e57–67.

    Article  PubMed  CAS  Google Scholar 

  53. Rupp LJ, Schumann K, Roybal KT, Gate RE, Ye CJ, Lim WA, et al. CRISPR/cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor t cells. Sci Rep. 2017;7:737.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to the staff in Biobank of Zhongda Hospital Affiliated to Southeast University for technical assistance. The authors would like to thank Shengmaiyi (Nanjing) Biotechnology Co. for providing infrastructure and financial support.

Author contributors

FJ, YS: Conception and design; FJ, KY, BD, YZ: Acquisition of data; FJ, MR, KG, HL, SL: Analysis and interpretation of data; FJ, KG, YS, KY: Writing, review, and revision of the manuscript; FJ, BD, YS: Administrative, technical, or material support; ZX, YS: Study supervision. All authors have read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (82303764, 82372126, and 82072078), the China Postdoctoral Science Foundation (2023M740616, 2025T180583, and 2024M750460), Young Elite Scientists Sponsorship Program by Jiangsu Association for Science and Technology (JSTJ-2025-502), the Beijing Xisike Clinical Oncology Research Foundation (Y-Young2024-0102, Y-zai2022/ms-0126), Noncommunicable Chronic Diseases-National Science and Technology Major Project (2025ZD0545600), the Research Talent Cultivation Program of Zhongda Hospital Affiliated to Southeast University (CZXM-GSP-RC06), and Jiangsu Province High-Level Hospital Pairing Assistance Construction Funds from Zhongda Hospital Affiliated to Southeast University (zdyyxy35).

Author information

Author notes

  1. These authors contributed equally: Feng Ji, Kai Yan, Bo Ding.

Authors and Affiliations

  1. Department of Obstetrics and Gynecology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, Jiangsu, China

    Feng Ji, Bo Ding, Yuxin Zhu, Mengchen Rao, Kexing Gao, Hao Lin, Yiyang Shan, Sicong Liu, Zhongdang Xiao & Yang Shen

  2. Department of Clinical Science and Research, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, Jiangsu, China

    Feng Ji & Hao Lin

  3. Nanjing Key Laboratory of Intelligent Obstetrics and Gynecology Medicine, Southeast University, Nanjing, Jiangsu, China

    Feng Ji, Bo Ding, Yuxin Zhu, Mengchen Rao, Hao Lin, Yiyang Shan, Sicong Liu & Yang Shen

  4. State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University, Nanjing, Jiangsu, China

    Kai Yan & Zhongdang Xiao

Authors
  1. Feng Ji
    View author publications

    Search author on:PubMed Google Scholar

  2. Kai Yan
    View author publications

    Search author on:PubMed Google Scholar

  3. Bo Ding
    View author publications

    Search author on:PubMed Google Scholar

  4. Yuxin Zhu
    View author publications

    Search author on:PubMed Google Scholar

  5. Mengchen Rao
    View author publications

    Search author on:PubMed Google Scholar

  6. Kexing Gao
    View author publications

    Search author on:PubMed Google Scholar

  7. Hao Lin
    View author publications

    Search author on:PubMed Google Scholar

  8. Yiyang Shan
    View author publications

    Search author on:PubMed Google Scholar

  9. Sicong Liu
    View author publications

    Search author on:PubMed Google Scholar

  10. Zhongdang Xiao
    View author publications

    Search author on:PubMed Google Scholar

  11. Yang Shen
    View author publications

    Search author on:PubMed Google Scholar

Corresponding authors

Correspondence to Zhongdang Xiao or Yang Shen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

All methods were performed in accordance with the relevant guidelines and regulations. All animal experiments were approved by the Institutional Animal Care and Use Committee of Southeast University (approval number 20230313001), in accordance with institutional guidelines. No human subjects were involved in this study.

Additional information

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

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ji, F., Yan, K., Ding, B. et al. Dual-target CAR-T therapy for ovarian cancer: synergistic targeting of MSLN and B7H3 enhances anti-tumor efficacy and overcomes antigen heterogeneity. Oncogene 45, 446–458 (2026). https://doi.org/10.1038/s41388-025-03663-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41388-025-03663-y

Search

Advanced search

Quick links