Skip to main content

Thank you for visiting 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:

Translational Therapeutics

Biparatopic anti-HER2 drug radioconjugates as breast cancer theranostics



HER2 is overexpressed in 25–30% of breast cancer. Multiple domains targeting of a receptor can have synergistic/additive therapeutic effects.


Two domain-specific ADCs trastuzumab-PEG6-DM1 (domain IV) and pertuzumab-PEG6-DM1 (domain II) were developed, characterised and radiolabeled to obtain [89Zr]Zr-trastuzumab-PEG6-DM1 and [67Cu]Cu-pertuzumab-PEG6-DM1 to study their in vitro (binding assay, internalisation and cytotoxicity) and in vivo (pharmacokinetics, biodistribution and immunoPET/SPECT imaging) characteristics.


The ADCs had an average drug-to-antibody ratio of 3. Trastuzumab did not compete with [67Cu]Cu-pertuzumab-PEG6-DM1 for binding to HER2. The highest antibody internalisation was observed with the combination of ADCs in BT-474 cells compared with single antibodies or ADCs. The combination of the two ADCs had the lowest IC50 compared with treatment using the single ADCs or controls. Pharmacokinetics showed biphasic half-lives with fast distribution and slow elimination, and an AUC that was five-fold higher for [89Zr]Zr-trastuzumab-PEG6-DM1 compared with [67Cu]Cu-pertuzumab-PEG6-DM1. Tumour uptake of [89Zr]Zr-trastuzumab-PEG6-DM1 was 51.3 ± 17.3% IA/g (BT-474), and 12.9 ± 2.1% IA/g (JIMT-1) which was similarly to [67Cu]Cu-pertuzumab-PEG6-DM1. Mice pre-blocked with pertuzumab had [89Zr]Zr-trastuzumab-PEG6-DM1 tumour uptakes of 66.3 ± 33.9% IA/g (BT-474) and 25.3 ± 4.9% IA/g (JIMT-1) at 120 h p.i.


Using these biologics simultaneously as biparatopic theranostic agents has additive benefits.

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

Access options

Buy this article

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

Fig. 1: Flow cytometry saturation binding assay of antibodies and immunoconjugates in HER2-positive BT-474 cells.
Fig. 2: Radioligand and competitive binding assays in HER2-positive BT-474 cells.
Fig. 3: Antibody internalisation of trastuzumab, trastuzumab-PEG6-DM1, pertuzumab, pertuzumab-PEG6-DM1 and a combination of trastuzumab-PEG6-DM1 + pertuzumab-PEG6-DM1 in HER2-positive BT-474 cell line after 48 h of incubation.
Fig. 4: In vitro cytotoxicity of immunoconjugates in cells with different HER2 densities.
Fig. 5: MicroPET/SPECT/CT and biodistribution of antibody–drug radioconjugates at different timepoints post injection.
Fig. 6: Targeted antibody drug radioconjugate-specific imaging of HER2-positive xenografts following simultaneous injection of [89Zr]Zr-trastuzumab-PEG6-DM1 + [67Cu]Cu-pertuzumab-PEG6-DM1.

Similar content being viewed by others

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.


  1. Canadian Cancer Statistics Advisory Committee in collaboration with the Canadian Cancer Society SCatPHAoC. Canadian Cancer Statistics 2021; 2021.

  2. Latta EK, Tjan S, Parkes RK, O’Malley FP. The role of HER2/neu overexpression/amplification in the progression of ductal carcinoma in situ to invasive carcinoma of the breast. Mod Pathol. 2002;15:1318–25.

    Article  CAS  PubMed  Google Scholar 

  3. Gote V, Nookala AR, Bolla PK, Pal D. Drug resistance in metastatic breast cancer: tumor targeted nanomedicine to the rescue. Int J Mol Sci. 2021;22:4673.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med. 2001;344:783–92.

    Article  CAS  PubMed  Google Scholar 

  5. Jahanzeb M. Adjuvant trastuzumab therapy for HER2-positive breast cancer. Clin Breast Cancer. 2008;8:324–33.

    Article  CAS  PubMed  Google Scholar 

  6. Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol. 2007;25:118–45.

    Article  CAS  PubMed  Google Scholar 

  7. Harris LN, You F, Schnitt SJ, Witkiewicz A, Lu X, Sgroi D, et al. Predictors of resistance to preoperative trastuzumab and vinorelbine for HER2-positive early breast cancer. Clin Cancer Res. 2007;13:1198–207.

    Article  CAS  PubMed  Google Scholar 

  8. Loibl S, Jackisch C, Schneeweiss A, Schmatloch S, Aktas B, Denkert C, et al. Dual HER2-blockade with pertuzumab and trastuzumab in HER2-positive early breast cancer: a subanalysis of data from the randomized phase III GeparSepto trial. Ann Oncol. 2017;28:497–504.

    Article  CAS  PubMed  Google Scholar 

  9. von Minckwitz G, Procter M, de Azambuja E, Zardavas D, Benyunes M, Viale G, et al. Adjuvant pertuzumab and trastuzumab in early HER2-positive breast cancer. N Engl J Med. 2017;377:122–31.

    Article  Google Scholar 

  10. Nami B, Maadi H, Wang Z. Mechanisms underlying the action and synergism of trastuzumab and pertuzumab in targeting HER2-positive breast cancer. Cancers. 2018;10:342.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Tsao L-C, Crosby EJ, Trotter TN, Wei J, Wang T, Yang X, et al. Trastuzumab/pertuzumab combination therapy stimulates antitumor responses through complement-dependent cytotoxicity and phagocytosis. JCI Insight. 2022;7:e155636.

  12. Richard S, Selle F, Lotz J-P, Khalil A, Gligorov J, Soares DG. Pertuzumab and trastuzumab: the rationale way to synergy. An da Academia Brasileira de Ciências. 2016;88:565–77.

    Article  CAS  Google Scholar 

  13. Verma S, Miles D, Gianni L, Krop IE, Welslau M, Baselga J, et al. Trastuzumab emtansine for HER2-positive advanced breast cancer. N Engl J Med. 2012;367:1783–91.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E, et al. Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res. 2008;68:9280–90.

    Article  CAS  PubMed  Google Scholar 

  15. Junutula JR, Flagella KM, Graham RA, Parsons KL, Ha E, Raab H, et al. Engineered thio-trastuzumab-DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer. Clin Cancer Res. 2010;16:4769–78.

    Article  CAS  PubMed  Google Scholar 

  16. Barok M, Joensuu H, Isola J. Trastuzumab emtansine: mechanisms of action and drug resistance. Breast Cancer Res. 2014;16:209.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Shefet-Carasso L, Benhar I. Antibody-targeted drugs and drug resistance-challenges and solutions. Drug Resist Updat. 2015;18:36–46.

    Article  PubMed  Google Scholar 

  18. Singh AP, Sharma S, Shah DK. Quantitative characterization of in vitro bystander effect of antibody-drug conjugates. J Pharmacokinet Pharmacodyn. 2016;43:567–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Modi S, Saura C, Yamashita T, Park YH, Kim SB, Tamura K, et al. Trastuzumab deruxtecan in previously treated HER2-positive breast cancer. N Engl J Med. 2020;382:610–21.

    Article  CAS  PubMed  Google Scholar 

  20. Keam SJ. Trastuzumab deruxtecan: first approval. Drugs. 2020;80:501–8.

    Article  CAS  PubMed  Google Scholar 

  21. 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 

  22. 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 

  23. Comer F, Gao CS, Coats S. Bispecific and biparatopic antibody drug conjugates. Cancer Drug Discov D. Cham; Springer International Publishing. 2018. p. 267–80.

  24. Hamblett KJ, Barnscher SD, Davies RH, Hammond PW, Hernandez A, Wickman GR, et al. ZW49, a HER2 targeted biparatopic antibody drug conjugate for the treatment of HER2 expressing cancers. Cancer Res. 2018;78:3914.

  25. Kovtun YV, Audette CA, Mayo MF, Jones GE, Doherty H, Maloney EK, et al. Antibody-maytansinoid conjugates designed to bypass multidrug resistance. Cancer Res. 2010;70:2528–37.

  26. Zhao RY, Wilhelm SD, Audette C, Jones G, Leece BA, Lazar AC, et al. Synthesis and evaluation of hydrophilic linkers for antibody-maytansinoid conjugates. J Med Chem. 2011;54:3606–23.

  27. Henry KE, Ulaner GA, Lewis JS. Human epidermal growth factor receptor 2-targeted PET/single-photon emission computed tomography imaging of breast cancer: noninvasive measurement of a biomarker integral to tumor treatment and prognosis. PET Clin. 2017;12:269–88.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Keinänen O, Fung K, Brennan JM, Zia N, Harris M, Van Dam E, et al. Harnessing64Cu/67Cu for a theranostic approach to pretargeted radioimmunotherapy. Proc Natl Acad Sci USA. 2020;117:28316–27.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Hao G, Mastren T, Silvers W, Hassan G, Öz OK, Sun X. Copper-67 radioimmunotheranostics for simultaneous immunotherapy and immuno-SPECT. Sci Rep. 2021;11:1–11.

  30. Liang Y, Besch-Williford C, Hyder SM. PRIMA-1 inhibits growth of breast cancer cells by re-activating mutant p53 protein. Int J Oncol. 2009;35:1015–23.

  31. Hartimath SV, Alizadeh E, Solomon VR, Chekol R, Bernhard W, Hill W, et al. Preclinical evaluation of (111)In-labeled PEGylated maytansine nimotuzumab drug conjugates in EGFR-positive cancer models. J Nucl Med. 2019;60:1103–10.

    Article  CAS  PubMed  Google Scholar 

  32. Hartimath SV, El-Sayed A, Makhlouf A, Bernhard W, Gonzalez C, Hill W, et al. Therapeutic potential of nimotuzumab PEGylated-maytansine antibody drug conjugates against EGFR positive xenograft. Oncotarget. 2019;10:1031–44.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Chen Y. Drug-to-antibody ratio (DAR) by UV/Vis spectroscopy. Totowa, NJ: Humana Press; 2013. p. 267–73.

  34. Tikum AF, Nambisan AK, Ketchemen JP, Babeker H, Khan MN, Torlakovic EE, et al. Simultaneous imaging and therapy using epitope-specific anti-epidermal growth factor receptor (EGFR) antibody conjugates. Pharmaceutics. 2022;14:1917.

  35. Solomon VR, Barreto K, Bernhard W, Alizadeh E, Causey P, Perron R, et al. Nimotuzumab site-specifically labeled with 89Zr and 225Ac using SpyTag/SpyCatcher for PET imaging and alpha particle radioimmunotherapy of epidermal growth factor receptor positive cancers. Cancers. 2020;12:3449.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Alizadeh E, Behlol Ayaz Ahmed K, Raja Solomon V, Gaja V, Bernhard W, Makhlouf A, et al. (89)Zr-labeled domain II-specific scFv-Fc immunoPET probe for imaging epidermal growth factor receptor in vivo. Cancers. 2021;13:560.

  37. Chekol R, Solomon VR, Alizadeh E, Bernhard W, Fisher D, Hill W, et al. (89)Zr-nimotuzumab for immunoPET imaging of epidermal growth factor receptor I. Oncotarget. 2018;9:17117–32.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Ferrario C, Christofides A, Joy AA, Laing K, Gelmon K, Brezden-Masley C. Novel therapies for the treatment of HER2-positive advanced breast cancer: a Canadian perspective. Curr Oncol. 2022;29:2720–34.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Swain SM, Baselga J, Kim S-B, Ro J, Semiglazov V, Campone M, et al. Pertuzumab, trastuzumab, and docetaxel in HER2-positive metastatic breast cancer. N Engl J Med. 2015;372:724–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Zhang X, Chen J, Weng Z, Li Q, Zhao L, Yu N, et al. A new anti-HER2 antibody that enhances the anti-tumor efficacy of trastuzumab and pertuzumab with a distinct mechanism of action. Mol Immunol. 2020;119:48–58.

    Article  CAS  PubMed  Google Scholar 

  41. Vernieri C, Milano M, Brambilla M, Mennitto A, Maggi C, Cona MS, et al. Resistance mechanisms to anti-HER2 therapies in HER2-positive breast cancer: current knowledge, new research directions and therapeutic perspectives. Crit Rev Oncol/Hematol. 2019;139:53–66.

    Article  PubMed  Google Scholar 

  42. Luque-Bolivar A, Pérez-Mora E, Villegas VE, Rondón-Lagos M. Resistance and overcoming resistance in breast cancer. Breast Cancer: Targets Ther. 2020;12:211–29.

  43. Hunter FW, Barker HR, Lipert B, Rothé 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 

  44. Veronese FM, Mero A. The impact of PEGylation on biological therapies. BioDrugs. 2008;22:315–29.

    Article  CAS  PubMed  Google Scholar 

  45. Massicano AVF, Lee S, Crenshaw BK, Aweda TA, El Sayed R, Super I, et al. Imaging of HER2 with [(89)Zr]pertuzumab in response to T-DM1 therapy. Cancer Biother Radiopharm. 2019;34:209–17.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Lam K, Chan C, Reilly RM. Development and preclinical studies of 64Cu-NOTA-pertuzumab F(ab′)2 for imaging changes in tumor HER2 expression associated with response to trastuzumab by PET/CT. mAbs. 2017;9:154–64.

    Article  CAS  PubMed  Google Scholar 

  47. Woo S-K, Jang SJ, Seo M-J, Park JH, Kim BS, Kim EJ, et al. Development of 64Cu-NOTA-trastuzumab for HER2 targeting: a radiopharmaceutical with improved pharmacokinetics for human studies. J Nucl Med. 2019;60:26–33.

    Article  CAS  PubMed  Google Scholar 

  48. Nair SK, Verma A, Thomas TJ, Chou TC, Gallo MA, Shirahata A, et al. Synergistic apoptosis of MCF-7 breast cancer cells by 2-methoxyestradiol and bis(ethyl)norspermine. Cancer Lett. 2007;250:311–22.

    Article  CAS  PubMed  Google Scholar 

  49. Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharm Rev. 2006;58:621–81.

    Article  CAS  PubMed  Google Scholar 

  50. Sanchez-Martin FJ, Bellosillo B, Gelabert-Baldrich M, Dalmases A, Canadas I, Vidal J, et al. The first-in-class anti-EGFR antibody mixture Sym004 overcomes cetuximab resistance mediated by EGFR extracellular domain mutations in colorectal cancer. Clin Cancer Res. 2016;22:3260–7.

    Article  PubMed  Google Scholar 

  51. Montagut C, Argiles G, Ciardiello F, Poulsen TT, Dienstmann R, Kragh M, et al. Efficacy of Sym004 in patients with metastatic colorectal cancer with acquired resistance to anti-EGFR therapy and molecularly selected by circulating tumor DNA analyses: a phase 2 randomized clinical trial. JAMA Oncol. 2018;4:e175245.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Al-Saden N, Lam K, Chan C, Reilly RM. Positron-emission tomography of HER2-positive breast cancer xenografts in mice with 89Zr-labeled trastuzumab-DM1: a comparison with 89Zr-labeled trastuzumab. Mol Pharm. 2018;15:3383–93.

    Article  CAS  PubMed  Google Scholar 

  53. Marquez BV, Ikotun OF, Zheleznyak A, Wright B, Hari-Raj A, Pierce RA, et al. Evaluation of (89)Zr-pertuzumab in Breast cancer xenografts. Mol Pharm. 2014;11:3988–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references


The authors wish to acknowledge the contributions of Dr. Vijay Gaja (Canadian Isotope Innovations Corp CIIC) for providing [67Cu]CuCl2, and Dr. Musharraf Khan (Saskatchewan Cyclotron Facility) for assistance with experiments.


This work was funded by Canadian Institute for Health Research (CIHR) Project Grants (# 437660 and 408132) to HF.

Author information

Authors and Affiliations



Experimental design, execution and data analysis were performed by JPK, HB, AFT, AKN, FNN, EN and HF. Writing of the original draft preparation and review were done by JPK and HF. All the authors contributed to the article and approved the submitted version.

Corresponding author

Correspondence to Humphrey Fonge.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

All animal experiments were approved, supervised, and maintained following the guidelines of the University of Saskatchewan Animal Care Committee (UACC). Ethical approval references 20170084 and 20220021.

Consent for publication

Not applicable.

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

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ketchemen, J.P., Babeker, H., Tikum, A.F. et al. Biparatopic anti-HER2 drug radioconjugates as breast cancer theranostics. Br J Cancer 129, 153–162 (2023).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links