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:

Novel anti-HER2 nanobody-drug conjugates with enhanced penetration of solid tumor and BBB, reduced systemic exposure and superior antitumor efficacy

Acta Pharmacologica Sinica volume 47, pages 467–480 (2026)Cite this article

Abstract

Antibody-drug conjugate (ADC) represents a promising paradigm for tumor-targeted delivery of chemotherapy. Trastuzumab deruxtecan (T-Dxd/DS-8201), a second-generation HER2-ADC, has significantly improved treatment outcomes for breast cancer patients. But due to the large molecular weight, the performance of ADC is still limited by lower tumor penetration, insufficient BBB permeability, and prolonged systemic exposure to normal tissues. In this study, we generated novel anti-HER2 nanobodies (VHH2, VHH3) that exhibited outstanding target affinity and tumor inhibition. After i.v. injection, VHH3-Fc fusion distributed 4 to 5-fold higher in subcutaneous tumor and intracranial tumor compared with trastuzumab. VHH3-Fc and VHH3-ABD were also more penetrant in an in vitro BBB permeability assay. Site-specific conjugation of VHH3-Fc or VHH3-ABD fusions with anti-microtubule MMAE or anti-topoisomerase-1 Dxd payload produced nanobody-drug conjugates (NDCs) with highly potent and durable antitumor efficacy. When evaluated on the same linker-payload (GGFG-Dxd) dosages, VHH3-Fc-Dxd (DAR3.9) outperformed T-Dxd (DAR8) in both the subcutaneous and intracranial tumor models. Moreover, IHC staining and RNA-seq analysis of the treated tumor tissues revealed the involvement of the cGAS-STING-IFNs pathway in mediating the drug activity. Gene expression and protein function were more profoundly modulated by VHH3-Fc-Dxd than T-Dxd. Unlike the higher tumor distribution, the mouse serum PK study revealed a faster clearance (T1/2), reduced exposure (AUC), and higher volume distribution (Vz) for VHH3-Fc-Dxd relative to T-Dxd. Our results provide an example for the next generation HER2-NDC with substantially differentiated pharmacokinetics and pharmacodynamics profiles that will further benefit treatment outcomes and therapeutic windows.

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: Generation and binding affinity of novel anti-HER2 nanobodies.
Fig. 2: Internalization and antitumor activity of the anti-HER2 nanobody.
Fig. 3: In vitro and in vivo efficacy of anti-HER2 nanobody-drug conjugates (NDCs).
Fig. 4: Biodistribution and tumor inhibition of HER2-NDC.
Fig. 5: In vivo antitumor efficacy mechanism of HER2-NDC.
Fig. 6: The biodistribution, intracranial tumor inhibition and BBB penetration of HER2-NDC.
Fig. 7: Novel HER2-NDCs exhibit better prospects for cancer therapy.

Similar content being viewed by others

Data availability

All relevant data are within the manuscript files and are available upon request.

References

  1. Yoon J, Oh DY. HER2-targeted therapies beyond breast cancer - an update. Nature reviews. Clin Oncol. 2024;21:675–700.

    CAS  Google Scholar 

  2. Cronin KA, Harlan LC, Dodd KW, Abrams JS, Ballard-Barbash R. Population-based estimate of the prevalence of HER-2 positive breast cancer tumors for early stage patients in the US. Cancer Invest. 2010;28:963–8.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Van Cutsem E, Bang YJ, Feng-Yi F, Xu JM, Lee KW, Jiao SC, et al. HER2 screening data from ToGA: targeting HER2 in gastric and gastroesophageal junction cancer. Gastric Cancer. 2015;18:476–84.

    Article  PubMed  Google Scholar 

  4. Raghav K, Siena S, Takashima A, Kato T, Van den Eynde M, Pietrantonio F, et al. Trastuzumab deruxtecan in patients with HER2-positive advanced colorectal cancer (DESTINY-CRC02): primary results from a multicentre, randomised, phase 2 trial. Lancet Oncol. 2024;25:1147–62.

    Article  CAS  PubMed  Google Scholar 

  5. Gutierrez C, Schiff R. HER2: biology, detection, and clinical implications. Arch Pathol Lab Med. 2011;135:55–62.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Madson JG, Lynch DT, Svoboda J, Ophardt R, Yanagida J, Putta SK, et al. Erbb2 suppresses DNA damage-induced checkpoint activation and UV-induced mouse skin tumorigenesis. Am J Pathol. 2009;174:2357–66.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Boone JJ, Bhosle J, Tilby MJ, Hartley JA, Hochhauser D. Involvement of the HER2 pathway in repair of DNA damage produced by chemotherapeutic agents. Mol Cancer Ther. 2009;8:3015–23.

    Article  CAS  PubMed  Google Scholar 

  8. Garcia-Rendueles MER, Krishnamoorthy G, Saqcena M, Acuna-Ruiz A, Revilla G, de Stanchina E, et al. Yap governs a lineage-specific neuregulin1 pathway-driven adaptive resistance to RAF kinase inhibitors. Mol Cancer. 2022;21:213.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kodack DP, Askoxylakis V, Ferraro GB, Sheng Q, Badeaux M, Goel S, et al. The brain microenvironment mediates resistance in luminal breast cancer to PI3K inhibition through HER3 activation. Sci Transl Med. 2017;9:eaal4682.

  10. Leonetti A, Sharma S, Minari R, Perego P, Giovannetti E, Tiseo M. Resistance mechanisms to osimertinib in EGFR-mutated non-small cell lung cancer. Br J Cancer. 2019;121:725–37.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Solanki HS, Welsh EA, Fang B, Izumi V, Darville L, Stone B, et al. Cell type-specific adaptive signaling responses to KRAS(G12C) Inhibition. Clin Cancer Res. 2021;27:2533–48.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Amiri-Kordestani L, Blumenthal GM, Xu QC, Zhang L, Tang SW, Ha L, et al. FDA approval: ado-trastuzumab emtansine for the treatment of patients with HER2-positive metastatic breast cancer. Clin Cancer Res. 2014;20:4436–41.

    Article  CAS  PubMed  Google Scholar 

  13. Hunter FW, Barker HR, Lipert B, Rothe F, Gebhart G, Piccart-Gebhart MJ, et al. Mechanisms of resistance to trastuzumab emtansine (T-DM1) in HER2-positive breast cancer. Br J Cancer. 2020;122:603–12.

    Article  CAS  PubMed  Google Scholar 

  14. Paz-Manrique R, Pinto JA, Gomez Moreno HL. Antibody-drug conjugates in breast cancer: toward a molecular perspective into clinical practice. JCO Precis Oncol. 2024;8:e2400173.

    Article  PubMed  Google Scholar 

  15. Swain SM, Shastry M, Hamilton E. Targeting HER2-positive breast cancer: advances and future directions. Nat Rev Drug Discov. 2023;22:101–26.

    Article  CAS  PubMed  Google Scholar 

  16. Colombo R, Rich JR. The therapeutic window of antibody drug conjugates: A dogma in need of revision. Cancer Cell. 2022;40:1255–63.

    Article  CAS  PubMed  Google Scholar 

  17. Guidi L, Pellizzari G, Tarantino P, Valenza C, Curigliano G. Resistance to antibody-drug conjugates targeting HER2 in breast cancer: molecular landscape and future challenges. Cancers. 2023;15:1130.

  18. Shitara K, Bang YJ, Iwasa S, Sugimoto N, Ryu MH, Sakai D, et al. Trastuzumab deruxtecan in HER2-positive advanced gastric cancer: exploratory biomarker analysis of the randomized, phase 2 DESTINY-Gastric01 trial. Nat Med. 2024;30:1933–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Maadi H, Soheilifar MH, Choi WS, Moshtaghian A, Wang Z. Trastuzumab mechanism of action; 20 years of research to unravel a dilemma. Cancers. 2021;13:3540.

  20. Abuhelwa Z, Alloghbi A, Alqahtani A, Nagasaka M. Trastuzumab Deruxtecan-induced interstitial lung disease/Pneumonitis in ERBB2-positive advanced solid malignancies: a systematic review. Drugs. 2022;82:979–87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Murrell DH, Foster PJ, Chambers AF. Brain metastases from breast cancer: lessons from experimental magnetic resonance imaging studies and clinical implications. J Mol Med. 2014;92:5–12.

    Article  PubMed  Google Scholar 

  22. Dasararaju R, Mehta A. Current advances in understanding and managing secondary brain metastasis. CNS Oncol. 2013;2:75–85.

    Article  CAS  PubMed  Google Scholar 

  23. Starkweather CK, Choi BD, Alvarez-Breckenridge C, Brastianos PK, Oh K, Wang N, et al. Initial approach to the patient with multiple newly diagnosed brain metastases. Neurosurg Clin N. Am. 2020;31:505–13.

    Article  PubMed  Google Scholar 

  24. Kennecke H, Yerushalmi R, Woods R, Cheang MC, Voduc D, Speers CH, et al. Metastatic behavior of breast cancer subtypes. J Clin Oncol. 2010;28:3271–7.

    Article  PubMed  Google Scholar 

  25. Chen PC, Yeh YM, Chu CT, Su PF, Chiu PH, Lin BW, et al. HER2 amplification in colorectal cancer with brain metastasis: A propensity score matching study. Eur J Cancer. 2023;181:62–9.

    Article  CAS  PubMed  Google Scholar 

  26. Lai MY, Guan WL, Yang J, Sun YT, Lu SX, Yang LQ, et al. The relationship between brain metastasis and HER2 expression status in gastric cancer. Clin Transl Oncol. 2024;26:765–73.

    Article  CAS  PubMed  Google Scholar 

  27. Kodack DP, Chung E, Yamashita H, Incio J, Duyverman AM, Song Y, et al. Combined targeting of HER2 and VEGFR2 for effective treatment of HER2-amplified breast cancer brain metastases. Proc Natl Acad Sci USA. 2012;109:E3119–27.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Montemurro F, Delaloge S, Barrios CH, Wuerstlein R, Anton A, Brain E, et al. Trastuzumab emtansine (T-DM1) in patients with HER2-positive metastatic breast cancer and brain metastases: exploratory final analysis of cohort 1 from KAMILLA, a single-arm phase IIIb clinical trial(☆). Ann Oncol. 2020;31:1350–8.

    Article  CAS  PubMed  Google Scholar 

  29. Michelon I, Vilbert M, Marinho AD, Castro CER, Dacoregio MI, Stecca C, et al. Trastuzumab deruxtecan in human epidermal growth factor receptor 2-positive breast cancer brain metastases: a systematic review and meta-analysis. ESMO Open. 2024;9:102233.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Olson EM, Abdel-Rasoul M, Maly J, Wu CS, Lin NU, Shapiro CL. Incidence and risk of central nervous system metastases as site of first recurrence in patients with HER2-positive breast cancer treated with adjuvant trastuzumab. Ann Oncol. 2013;24:1526–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Lewis Phillips GD, Nishimura MC, Lacap JA, Kharbanda S, Mai E, Tien J, et al. Trastuzumab uptake and its relation to efficacy in an animal model of HER2-positive breast cancer brain metastasis. Breast Cancer Res Treat. 2017;164:581–91.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Hurvitz SA, Kim SB, Chung WP, Im SA, Park YH, Hegg R, et al. Trastuzumab deruxtecan versus trastuzumab emtansine in HER2-positive metastatic breast cancer patients with brain metastases from the randomized DESTINY-Breast03 trial. ESMO Open. 2024;9:102924.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ogitani Y, Hagihara K, Oitate M, Naito H, Agatsuma T. Bystander killing effect of DS-8201a, a novel anti-human epidermal growth factor receptor 2 antibody-drug conjugate, in tumors with human epidermal growth factor receptor 2 heterogeneity. Cancer Sci. 2016;107:1039–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Nessler I, Khera E, Vance S, Kopp A, Qiu Q, Keating TA, et al. Increased tumor penetration of single-domain antibody-drug conjugates improves in vivo efficacy in prostate cancer models. Cancer Res. 2020;80:1268–78.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Bannas P, Lenz A, Kunick V, Fumey W, Rissiek B, Schmid J, et al. Validation of nanobody and antibody based in vivo tumor xenograft NIRF-imaging experiments in mice using ex vivo flow cytometry and microscopy. J Vis Exp 2015;98:e52462.

  36. Rutgers KS, Nabuurs RJ, van den Berg SA, Schenk GJ, Rotman M, Verrips CT, et al. Transmigration of beta amyloid specific heavy chain antibody fragments across the in vitro blood-brain barrier. Neuroscience. 2011;190:37–42.

    Article  CAS  PubMed  Google Scholar 

  37. Li T, Bourgeois JP, Celli S, Glacial F, Le Sourd AM, Mecheri S, et al. Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging. FASEB J. 2012;26:3969–79.

    Article  CAS  PubMed  Google Scholar 

  38. Jin BK, Odongo S, Radwanska M, Magez S. NANOBODIES®: A review of diagnostic and therapeutic applications. Int J Mol Sci. 2023;24:5994.

  39. Li JY, Perry SR, Muniz-Medina V, Wang X, Wetzel LK, Rebelatto MC, et al. A biparatopic HER2-targeting antibody-drug conjugate induces tumor regression in primary models refractory to or ineligible for HER2-targeted therapy. Cancer Cell. 2016;29:117–29.

    Article  CAS  PubMed  Google Scholar 

  40. Shi D, Mi G, Shen Y, Webster TJ. Glioma-targeted dual functionalized thermosensitive Ferri-liposomes for drug delivery through an in vitro blood-brain barrier. Nanoscale. 2019;11:15057–71.

    Article  CAS  PubMed  Google Scholar 

  41. Zakharova M, Tibbe MP, Koch LS, Le‐The H, Leferink AM, van den Berg A, et al. Transwell‐integrated 2 µm thick transparent polydimethylsiloxane membranes with controlled pore sizes and distribution to model the blood‐brain barrier. Adv Mater Technol. 2021;6:2100138.

  42. Bell A, Wang ZJ, Arbabi-Ghahroudi M, Chang TA, Durocher Y, Trojahn U, et al. Differential tumor-targeting abilities of three single-domain antibody formats. Cancer Lett. 2010;289:81–90.

    Article  CAS  PubMed  Google Scholar 

  43. Sleep D, Cameron J, Evans LR. Albumin as a versatile platform for drug half-life extension. Biochim Biophys Acta. 2013;1830:5526–34.

    Article  CAS  PubMed  Google Scholar 

  44. Chiu ML, Goulet DR, Teplyakov A, Gilliland GL. Antibody structure and function: the basis for engineering therapeutics. Antibodies. 2019;8:55.

  45. Sun D, Luo T, Dong P, Zhang N, Chen J, Zhang S, et al. CD86+/CD206+ tumor-associated macrophages predict prognosis of patients with intrahepatic cholangiocarcinoma. PeerJ. 2020;8:e8458.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Pepin G, Nejad C, Ferrand J, Thomas BJ, Stunden HJ, Sanij E, et al. Topoisomerase 1 inhibition promotes cyclic GMP-AMP Synthase-dependent antiviral responses. mBio. 2017;8:e01611–17.

  47. Marinello J, Arleo A, Russo M, Delcuratolo M, Ciccarelli F, Pommier Y, et al. Topoisomerase I poison-triggered immune gene activation is markedly reduced in human small-cell lung cancers by impairment of the cGAS/STING pathway. Br J Cancer. 2022;127:1214–25.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Wu S, Zhang Q, Zhang F, Meng F, Liu S, Zhou R, et al. HER2 recruits AKT1 to disrupt STING signalling and suppress antiviral defence and antitumour immunity. Nat Cell Biol. 2019;21:1027–40.

    Article  CAS  PubMed  Google Scholar 

  49. Wu Y, Li Q, Kong Y, Wang Z, Lei C, Li J, et al. A highly stable human single-domain antibody-drug conjugate exhibits superior penetration and treatment of solid tumors. Mol Ther. 2022;30:2785–99.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Xu C, Zhu M, Wang Q, Cui J, Huang Y, Huang X, et al. TROP2-directed nanobody-drug conjugate elicited potent antitumor effect in pancreatic cancer. J Nanobiotechnol. 2023;21:410.

    Article  CAS  Google Scholar 

  51. Loganzo F, Sung M, Gerber HP. Mechanisms of resistance to antibody-drug conjugates. Mol Cancer Ther. 2016;15:2825–34.

    Article  CAS  PubMed  Google Scholar 

  52. Xia T, Konno H, Ahn J, Barber GN. Deregulation of STING signaling in colorectal carcinoma constrains DNA damage responses and correlates with tumorigenesis. Cell Rep. 2016;14:282–97.

    Article  CAS  PubMed  Google Scholar 

  53. Falahat R, Perez-Villarroel P, Mailloux AW, Zhu G, Pilon-Thomas S, Barber GN, et al. STING signaling in melanoma cells shapes antigenicity and can promote antitumor T-cell activity. Cancer Immunol Res. 2019;7:1837–48.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Oh KS, Nam AR, Bang JH, Jeong Y, Choo SY, Kim HJ, et al. Immunomodulatory effects of trastuzumab deruxtecan through the cGAS-STING pathway in gastric cancer cells. Cell Commun Signal. 2024;22:518.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Alahmari A. Blood-brain barrier overview: structural and functional correlation. Neural Plast. 2021;2021:6564585.

    Article  PubMed  PubMed Central  Google Scholar 

  56. Parodi A, Rudzinska M, Deviatkin AA, Soond SM, Baldin AV, Zamyatnin AA, Jr. Established and emerging strategies for drug delivery across the blood-brain barrier in brain cancer. Pharmaceutics. 2019;11:245.

Download references

Acknowledgements

This work was funded by Fudan University (EZF301002), National Natural Science Foundation of China (81373442), NST Major Project of China (2018ZX09711002-008) and NBR 973 Program of China (2013CB932500). The authors thank Animal Experiment Center and Instrument Center at Fudan School of Pharmacy for the technical support.

Author information

Authors and Affiliations

  1. Department of Pharmacology, Fudan University School of Pharmacy, Shanghai, 201203, China

    Yue Wang, Liang Liu, Qi-yu Yang & Ker Yu

  2. Shanghai Key Laboratory of Oncology Target Discovery and Antibody Drug Development, Shanghai, 201203, China

    Ker Yu

Authors
  1. Yue Wang
    View author publications

    Search author on:PubMed Google Scholar

  2. Liang Liu
    View author publications

    Search author on:PubMed Google Scholar

  3. Qi-yu Yang
    View author publications

    Search author on:PubMed Google Scholar

  4. Ker Yu
    View author publications

    Search author on:PubMed Google Scholar

Contributions

YW: Conceptualization, Data curation, Formal analysis, Methodology, Project administration, Manuscript Writing. LL: Data curation, Formal analysis, Methodology. QYY: Visualization, Methodology. KY: Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Manuscript Writing.

Corresponding author

Correspondence to Ker Yu.

Ethics declarations

Competing interests

Y. Wang, L. Liu and K. Yu are inventors of a related patent application, international patent application number PCT/CN2023/101129. QY. Yang declares no conflict of interest.

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

Wang, Y., Liu, L., Yang, Qy. et al. Novel anti-HER2 nanobody-drug conjugates with enhanced penetration of solid tumor and BBB, reduced systemic exposure and superior antitumor efficacy. Acta Pharmacol Sin 47, 467–480 (2026). https://doi.org/10.1038/s41401-025-01634-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41401-025-01634-3

Keywords

Search

Advanced search

Quick links