Development and Evaluation of Tri-Functional Immunoliposomes for the Treatment of HER2 Positive Breast Cancer

  • Tanaya Vaidya
  • Robert M. Straubinger
  • Sihem Ait-Oudhia
Research Paper



Trastuzumab combined with Doxorubicin (DOX) demonstrates significant clinical activity in human epidermal growth factor receptor-2 (HER2)-positive breast cancer (BC). However, emergence of treatment resistance and trastuzumab associated cardiotoxicity remain clinical challenges. In an effort to improve patient outcome, we have developed and evaluated novel tri-functional immunoliposomes (TFIL) that target HER2-receptors on BC cells and CD3-receptors on T-lymphocytes, and deliver DOX.


Trastuzumab (anti-HER2) and OKT-3 (anti-CD3) antibodies were conjugated to liposomes using a micelle-transfer method. Cytotoxicity of targeted immunoliposomes loaded with DOX was examined in vitro on HER2-positive BC cells (BT474), with peripheral blood monocytic cells (PBMC) as immune effector cells.


TFIL demonstrated high antibody-liposome conjugation ratios (100–130 μg protein/μmol phospholipid) and cargo capacity (0.21 mol:mol drug:lipid), highly efficient DOX loading (>90%), a particle size favorable for extended circulation (~150 nm), and good stability (up to 3 months at 4°C). In the presence of PBMCs, TFIL showed complete killing of BT474 cells, and were superior to mono-targeted trastuzumab-bearing liposomes, non-targeted liposomes, and free Trastuzumab and DOX.


Novel anti-HER2xCD3 + DOX TFIL show promise as a means to both engage immune cells against HER2 positive breast cancer cells and deliver chemotherapy, and have the potential to improve clinical outcomes.


anti-CD3 HER2-positive breast cancer immunoliposomes trastuzumab doxorubicin 



Breast Cancer


Cluster of Differentiation Receptor Type 3


Cytotoxic T Lymphocytes




Human Epidermal Growth Factor Receptor Type 2


Peripheral Blood Mononuclear Cells


Tri-Functional Immunoliposomes


  1. 1.
    Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017. CA Cancer J Clin. 2017;67(1):7–30.CrossRefPubMedGoogle Scholar
  2. 2.
    Burstein HJ. The distinctive nature of HER2-positive breast cancers. N Engl J Med. 2005;353(16):1652–4.CrossRefPubMedGoogle Scholar
  3. 3.
    Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science (New York). 1987;235(4785):177–82.CrossRefGoogle Scholar
  4. 4.
    Harari D, Yarden Y. Molecular mechanisms underlying ErbB2/HER2 action in breast cancer. Oncogene. 2000;19(53):6102–14.CrossRefPubMedGoogle Scholar
  5. 5.
    Yarden Y. Biology of HER2 and its importance in breast cancer. Oncology. 2001;61(Suppl 2):1–13.CrossRefPubMedGoogle Scholar
  6. 6.
    Kallioniemi OP, Kallioniemi A, Kurisu W, Thor A, Chen LC, Smith HS, et al. ERBB2 amplification in breast cancer analyzed by fluorescence in situ hybridization. Proc Natl Acad Sci U S A. 1992;89(12):5321–5.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    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(20):3271–7.CrossRefPubMedGoogle Scholar
  8. 8.
    Valabrega G, Montemurro F, Aglietta M. Trastuzumab: mechanism of action, resistance and future perspectives in HER2-overexpressing breast cancer. Ann Oncol. 2007;18(6):977–84.CrossRefPubMedGoogle Scholar
  9. 9.
    Arteaga CL, Sliwkowski MX, Osborne CK, Perez EA, Puglisi F, Gianni L. Treatment of HER2-positive breast cancer: current status and future perspectives. Nat Rev Clin Oncol. 2012;9(1):16–32.CrossRefGoogle Scholar
  10. 10.
    Baselga J, Albanell J. Mechanism of action of anti-HER2 monoclonal antibodies. Ann Oncol. 2001;12(Suppl 1):S35–41.CrossRefPubMedGoogle Scholar
  11. 11.
    Petit AM, Rak J, Hung MC, Rockwell P, Goldstein N, Fendly B, et al. Neutralizing antibodies against epidermal growth factor and ErbB-2/neu receptor tyrosine kinases down-regulate vascular endothelial growth factor production by tumor cells in vitro and in vivo: angiogenic implications for signal transduction therapy of solid tumors. Am J Pathol. 1997;151(6):1523–30.PubMedPubMedCentralGoogle Scholar
  12. 12.
    Mohsin SK, Weiss HL, Gutierrez MC, Chamness GC, Schiff R, Digiovanna MP, et al. Neoadjuvant trastuzumab induces apoptosis in primary breast cancers. J Clin Oncol. 2005;23(11):2460–8.CrossRefPubMedGoogle Scholar
  13. 13.
    Baselga J. Clinical trials of Herceptin(R) (trastuzumab). Eur J Cancer. 2001;37(Suppl 1):18–24.CrossRefPubMedGoogle Scholar
  14. 14.
    Romond EH, Perez EA, Bryant J, Suman VJ, Geyer CE Jr, Davidson NE, et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med. 2005;353(16):1673–84.CrossRefPubMedGoogle Scholar
  15. 15.
    Slamon D, Eiermann W, Robert N, Pienkowski T, Martin M, Press M, et al. Adjuvant trastuzumab in HER2-positive breast cancer. N Engl J Med. 2011;365(14):1273–83.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L, et al. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol. 2002;20(3):719–26.CrossRefPubMedGoogle Scholar
  17. 17.
    Onitilo AA, Engel JM, Stankowski RV. Cardiovascular toxicity associated with adjuvant trastuzumab therapy: prevalence, patient characteristics, and risk factors. Ther Adv Drug Saf. 2014;5(4):154–66.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Pohlmann PR, Mayer IA, Mernaugh R. Resistance to Trastuzumab in breast cancer. Clin Cancer Res. 2009;15(24):7479–91.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Rexer BN, Arteaga CL. Intrinsic and acquired resistance to HER2-targeted therapies in HER2 gene-amplified breast cancer: mechanisms and clinical implications. Crit Rev Oncog. 2012;17(1):1–16.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Staerz UD, Kanagawa O, Bevan MJ. Hybrid antibodies can target sites for attack by T cells. Nature. 1985;314(6012):628–31.CrossRefPubMedGoogle Scholar
  21. 21.
    Perez P, Hoffman RW, Shaw S, Bluestone JA, Segal DM. Specific targeting of cytotoxic T cells by anti-T3 linked to anti-target cell antibody. Nature. 1985;316(6026):354–6.CrossRefPubMedGoogle Scholar
  22. 22.
    O'Brien ME, Wigler N, Inbar M, Rosso R, Grischke E, Santoro A, et al. Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYX/Doxil) versus conventional doxorubicin for first-line treatment of metastatic breast cancer. Ann Oncol. 2004;15(3):440–9.CrossRefPubMedGoogle Scholar
  23. 23.
    Torchilin V, Weissig V. Liposomes: a practical approach. New York: Oxford University Press; 2003.Google Scholar
  24. 24.
    Saito R, Bringas JR, McKnight TR, Wendland MF, Mamot C, Drummond DC, et al. Distribution of liposomes into brain and rat brain tumor models by convection-enhanced delivery monitored with magnetic resonance imaging. Cancer Res. 2004;64(7):2572–9.CrossRefPubMedGoogle Scholar
  25. 25.
    Abraham SA, Waterhouse DN, Mayer LD, Cullis PR, Madden TD, Bally MB. The liposomal formulation of doxorubicin. Methods Enzymol. 2005;391:71–97.CrossRefPubMedGoogle Scholar
  26. 26.
    Bartlett GR. Phosphorus assay in column chromatography. J Biol Chem. 1959;234(3):466–8.PubMedGoogle Scholar
  27. 27.
    Ishida T, Iden DL, Allen TM. A combinatorial approach to producing sterically stabilized (stealth) immunoliposomal drugs. FEBS Lett. 1999;460(1):129–33.CrossRefPubMedGoogle Scholar
  28. 28.
    Kastantin M, Ananthanarayanan B, Karmali P, Ruoslahti E, Tirrell M. Effect of the lipid chain melting transition on the stability of DSPE-PEG(2000) micelles. Langmuir. 2009;25(13):7279–86.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Heath TD, Macher BA, Papahadjopoulos D. Covalent attachment of immunoglobulins to liposomes via glycosphingolipids. Biochim Biophys Acta. 1981;640(1):66–81.CrossRefPubMedGoogle Scholar
  30. 30.
    Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, et al. New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst. 1990;82(13):1107–12.CrossRefPubMedGoogle Scholar
  31. 31.
    Holford NH, Sheiner LB. Kinetics of pharmacologic response. Pharmacol Ther. 1982;16(2):143–66.CrossRefPubMedGoogle Scholar
  32. 32.
    D’Argenio DZ, Schumitzky A, Wang X. user’s guide: pharmacokinetic/pharmacodynamics systems analysis software. Biomedical simulations resource, Los Angeles. 2009.Google Scholar
  33. 33.
    Lewis GD, Figari I, Fendly B, Wong WL, Carter P, Gorman C, et al. Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies. Cancer Immunol Immunother. 1993;37(4):255–63.CrossRefPubMedGoogle Scholar
  34. 34.
    Seidel UJ, Schlegel P, Lang P. Natural killer cell mediated antibody-dependent cellular cytotoxicity in tumor immunotherapy with therapeutic antibodies. Front Immunol. 2013;4:76.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Zitvogel L, Tesniere A, Apetoh L, Ghiringhelli F, Kroemer G. Immunological aspects of anticancer chemotherapy. Bull Acad Natl Med. 2008;192(7):1469–87. discussion 87-9PubMedGoogle Scholar
  36. 36.
    Smyth MJ, Thia KY, Street SE, MacGregor D, Godfrey DI, Trapani JA. Perforin-mediated cytotoxicity is critical for surveillance of spontaneous lymphoma. J Exp Med. 2000;192(5):755–60.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Kramer-Marek G, Kiesewetter DO, Capala J. Changes in HER2 expression in breast cancer xenografts after therapy can be quantified using PET and (18)F-labeled affibody molecules. J Nucl Med. 2009;50(7):1131–9.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Schlitt A, Jordan K, Vordermark D, Schwamborn J, Langer T, Thomssen C. Cardiotoxicity and oncological treatments. Dtsch Arztebl Int. 2014;111(10):161–8.PubMedPubMedCentralGoogle Scholar
  39. 39.
    Martin FJ, Papahadjopoulos D. Irreversible coupling of immunoglobulin fragments to preformed vesicles. An improved method for liposome targeting. J Biol Chem. 1982;257(1):286–8.PubMedGoogle Scholar
  40. 40.
    Mamot C, Drummond DC, Greiser U, Hong K, Kirpotin DB, Marks JD, et al. Epidermal growth factor receptor (EGFR)-targeted immunoliposomes mediate specific and efficient drug delivery to EGFR- and EGFRvIII-overexpressing tumor cells. Cancer Res. 2003;63(12):3154–61.PubMedGoogle Scholar
  41. 41.
    Marano N, Holowka D, Baird B. Bivalent binding of an anti-CD3 antibody to Jurkat cells induces association of the T cell receptor complex with the cytoskeleton. J Immunol. 1989;143(3):931–8.PubMedGoogle Scholar
  42. 42.
    Monjas A, Alcover A, Alarcón B. Engaged and bystander T cell receptors are down-modulated by different endocytotic pathways. J Biol Chem. 2004;279(53):55376–84.CrossRefPubMedGoogle Scholar
  43. 43.
    Landegren U, Andersson J, Wigzell H. Mechanism of T lymphocyte activation by OKT3 antibodies. A general model for T cell induction. Eur J Immunol. 1984;14(4):325–8.CrossRefPubMedGoogle Scholar
  44. 44.
    Gabizon A, Shmeeda H, Barenholz Y. Pharmacokinetics of pegylated liposomal doxorubicin: review of animal and human studies. Clin Pharmacokinet. 2003;42(5):419–36.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Tanaya Vaidya
    • 1
  • Robert M. Straubinger
    • 2
  • Sihem Ait-Oudhia
    • 1
  1. 1.Center for Pharmacometrics and Systems Pharmacology, Department of Pharmaceutics, College of PharmacyUniversity of FloridaOrlandoUSA
  2. 2.Department of Pharmaceutical SciencesUniversity at Buffalo, State University of New YorkBuffaloUSA

Personalised recommendations