Tumor delivery of antisense oligomer using trastuzumab within a streptavidin nanoparticle

Original Article

Abstract

Purpose

Trastuzumab (Herceptin™) is often internalized following binding to Her2+ tumor cells. The objective of this study was to investigate whether trastuzumab can be used as a specific carrier to deliver antisense oligomers into Her2+ tumor cells both in vitro and in vivo.

Methods

A biotinylated MORF oligomer antisense to RhoC mRNA and its biotinylated sense control were labeled with either lissamine for fluorescence detection or 99mTc for radioactivity detection and were linked to biotinylated trastuzumab via streptavidin. The nanoparticles were studied in SUM190 (RhoC+, Her2+) study and SUM149 (RhoC+, Her2−) control cells in culture and as xenografts in mice.

Results

As evidence of unimpaired Her2+ binding of trastuzumab within the nanoparticle, accumulations were clearly higher in SUM190 compared to SUM149 cells and, by whole-body imaging, targeting of SUM190 tumor was similar to that expected for a radiolabeled trastuzumab. As evidence of internalization, fluorescence microscopy images of cells grown in culture and obtained from xenografts showed uniform cytoplasm distribution of the lissamine-MORF. An invasion assay showed decreased RhoC expression in SUM190 cells when incubated with the antisense MORF nanoparticles at only 100 nM.

Conclusion

Both in cell culture and in animals, the nanoparticle with trastuzumab as specific carrier greatly improved tumor delivery of the antisense oligomer against RhoC mRNA into tumor cells overexpressing Her2 and may be of general utility.

Keywords

Antisense Trastuzumab Streptavidin MORF oligomer Nanoparticle 

Notes

Acknowledgements

The authors are grateful to Dr. Hendrik Pretorius, University of Massachusetts Medical School, for help with image quantitation, and to Dr. Kayoko Nakamura, Department of Radiology, Keio University School of Medicine, Tokyo 160-8582, Japan, for helpful discussion. This research was supported by the Congressionally Directed Medical Research Programs (CDMRP), grant No. W81XWH-06-1-0649 and in part the National Institutes of Health, grant No. SR21CA100092-02.

References

  1. 1.
    Nakamura K, Wang Y, Liu X, Kubo A, Hnatowich DJ. Cationic transfectors increase accumulation in cultured tumor cells of radiolabeled antisense DNAs without entrapment. Cancer Biother Radiopharm 2007;22:629–35.CrossRefPubMedGoogle Scholar
  2. 2.
    Zhang C, Tang N, Liu X, Liang W, Xu W, Torchilin VP. siRNA-containing liposomes modified with polyarginine effectively silence the targeted gene. J Control Release 2006;112:229–39.CrossRefPubMedGoogle Scholar
  3. 3.
    Breunig M, Hozsa C, Lungwitz U, Watanabe K, Umeda I, Kato H, et al. Mechanistic investigation of poly(ethylene imine)-based siRNA delivery: disulfide bonds boost intracellular release of the cargo. J Control Release 2008;130:57–63.CrossRefPubMedGoogle Scholar
  4. 4.
    Kim SH, Jeong JH, Lee SH, Kim SW, Park TG. Local and systemic delivery of VEGF siRNA using polyelectrolyte complex micelles for effective treatment of cancer. J Control Release 2008;129:107–16.CrossRefPubMedGoogle Scholar
  5. 5.
    de Fougerolles AR. Delivery vehicles for small interfering RNA in vivo. Hum Gene Ther 2008;19:125–32.CrossRefPubMedGoogle Scholar
  6. 6.
    Liu M, Wang RF, Zhang CL, Yan P, Yu MM, Di LJ, et al. Noninvasive imaging of human telomerase reverse transcriptase (hTERT) messenger RNA with 99mTc-radiolabeled antisense probes in malignant tumors. J Nucl Med 2007;48:2028–36.CrossRefPubMedGoogle Scholar
  7. 7.
    Nakamura K, Fan C, Liu G, Gupta S, He J, Dou S, et al. Evidence of antisense tumor targeting in mice. Bioconjug Chem 2004;15:1475–80.CrossRefPubMedGoogle Scholar
  8. 8.
    Liu N, Ding H, Vanderheyden JL, Zhu Z, Zhang Y. Radiolabeling small RNA with technetium-99 m for visualizing cellular delivery and mouse biodistribution. Nucl Med Biol 2007;34:399–404.CrossRefPubMedGoogle Scholar
  9. 9.
    Wang Y, Nakamura K, Liu X, Kitamura N, Kubo A, Hnatowich DJ. Simplified preparation via streptavidin of antisense oligomers/carriers nanoparticles showing improved cellular delivery in culture. Bioconjug Chem 2007;18:1338–43.CrossRefPubMedGoogle Scholar
  10. 10.
    Nakamura K, Wang Y, Liu X, Kubo A, Hnatowich DJ. Cell culture and xenograft-bearing animal studies of radiolabeled antisense DNA carrier nanoparticles with streptavidin as a linker. J Nucl Med 2007;48:1845–52.CrossRefPubMedGoogle Scholar
  11. 11.
    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;353:1673–84.CrossRefPubMedGoogle Scholar
  12. 12.
    Nahta R, Esteva FJ. HER-2-targeted therapy: lessons learned and future directions. Clin Cancer Res 2003;9:5078–84.PubMedGoogle Scholar
  13. 13.
    Piccart-Gebhart MJ, Procter M, Leyland-Jones B, Goldhirsch A, Untch M, Smith I, et al. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 2005;353:1659–72.CrossRefPubMedGoogle Scholar
  14. 14.
    Bange J, Zwick E, Ullrich A. Molecular targets for breast cancer therapy and prevention. Nat Med 2001;7:548–52.CrossRefPubMedGoogle Scholar
  15. 15.
    Perik PJ, Lub-De Hooge MN, Gietema JA, van der Graaf WT, de Korte MA, Jonkman S, et al. Indium-111-labeled trastuzumab scintigraphy in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer. J Clin Oncol 2006;24:2276–82.CrossRefPubMedGoogle Scholar
  16. 16.
    Sampath L, Kwon S, Ke S, Wang W, Schiff R, Mawad ME, et al. Dual-labeled trastuzumab-based imaging agent for the detection of human epidermal growth factor receptor 2 overexpression in breast cancer. J Nucl Med 2007;48:1501–10.CrossRefPubMedGoogle Scholar
  17. 17.
    Palm S, Enmon RM Jr, Matei C, Kolbert KS, Xu S, Zanzonico PB, et al. Pharmacokinetics and biodistribution of (86)Y-trastuzumab for (90)Y dosimetry in an ovarian carcinoma model: correlative MicroPET and MRI. J Nucl Med 2003;44:1148–55.PubMedGoogle Scholar
  18. 18.
    Kobayashi H, Shirakawa K, Kawamoto S, Saga T, Sato N, Hiraga A, et al. Rapid accumulation and internalization of radiolabeled herceptin in an inflammatory breast cancer xenograft with vasculogenic mimicry predicted by the contrast-enhanced dynamic MRI with the macromolecular contrast agent G6-(1B4M-Gd)(256). Cancer Res 2002;62:860–6.PubMedGoogle Scholar
  19. 19.
    Cho HS, Mason K, Ramyar KX, Stanley AM, Gabelli SB, Denney DW Jr, et al. Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab. Nature 2003;421:756–60.CrossRefPubMedGoogle Scholar
  20. 20.
    Mandler R, Kobayashi H, Hinson ER, Brechbiel MW, Waldmann TA. Herceptin-geldanamycin immunoconjugates: pharmacokinetics, biodistribution, and enhanced antitumor activity. Cancer Res 2004;64:1460–7.CrossRefPubMedGoogle Scholar
  21. 21.
    van Golen KL, Wu ZF, Qiao XT, Bao LW, Merajver SD. RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype. Cancer Res 2000;60:5832–8.PubMedGoogle Scholar
  22. 22.
    Clark EA, Golub TR, Lander ES, Hynes RO. Genomic analysis of metastasis reveals an essential role for RhoC. Nature 2000;406:532–5.CrossRefPubMedGoogle Scholar
  23. 23.
    van Golen KL, Bao LW, Pan Q, Miller FR, Wu ZF, Merajver SD. Mitogen activated protein kinase pathway is involved in RhoC GTPase induced motility, invasion and angiogenesis in inflammatory breast cancer. Clin Exp Metastasis 2002;19:301–11.CrossRefPubMedGoogle Scholar
  24. 24.
    Hakem A, Sanchez-Sweatman O, You-Ten A, Duncan G, Wakeham A, Khokha R, et al. RhoC is dispensable for embryogenesis and tumor initiation but essential for metastasis. Genes Dev 2005;19:1974–9.CrossRefPubMedGoogle Scholar
  25. 25.
    Kleer CG, Griffith KA, Sabel MS, Gallagher G, van Golen KL, Wu ZF, et al. RhoC-GTPase is a novel tissue biomarker associated with biologically aggressive carcinomas of the breast. Breast Cancer Res Treat 2005;93:101–10.CrossRefPubMedGoogle Scholar
  26. 26.
    Kleer CG, van Golen KL, Zhang Y, Wu ZF, Rubin MA, Merajver SD. Characterization of RhoC expression in benign and malignant breast disease: a potential new marker for small breast carcinomas with metastatic ability. Am J Pathol 2002;160:579–84.PubMedGoogle Scholar
  27. 27.
    Wang Y, Liu X, Hnatowich DJ. An improved synthesis of NHS-MAG3 for conjugation and radiolabeling of biomolecules with (99m)Tc at room temperature. Nat Protoc 2007;2:972–8.CrossRefPubMedGoogle Scholar
  28. 28.
    Wang Y, Liu G, Hnatowich DJ. Methods for MAG3 conjugation and 99mTc radiolabeling of biomolecules. Nat Protoc 2006;1:1477–80.CrossRefPubMedGoogle Scholar
  29. 29.
    Liu X, Wang Y, Nakamura K, Kawauchi S, Akalin A, Cheng D, et al. Auger radiation-induced, antisense-mediated cytotoxicity of tumor cells using a 3-component streptavidin-delivery nanoparticle with 111In. J Nucl Med 2009;50:582–90.CrossRefPubMedGoogle Scholar
  30. 30.
    Kandimalla VB, Neeta NS, Karanth NG, Thakur MS, Roshini KR, Rani BE, et al. Regeneration of ethyl parathion antibodies for repeated use in immunosensor: a study on dissociation of antigens from antibodies. Biosens Bioelectron 2004;20:903–6.CrossRefPubMedGoogle Scholar
  31. 31.
    Liu G, Dou S, Yin D, Squires S, Liu X, Wang Y, et al. A novel pretargeting method for measuring antibody internalization in tumor cells. Cancer Biother Radiopharm 2007;22:33–9.CrossRefPubMedGoogle Scholar
  32. 32.
    Etienne-Manneville S, Hall A. Rho GTPases in cell biology. Nature 2002;420:629–35.CrossRefPubMedGoogle Scholar
  33. 33.
    Liu X, Wang Y, Nakamura K, Kubo A, Hnatowich DJ. Cell studies of a three-component antisense MORF/tat/Herceptin nanoparticle designed for improved tumor delivery. Cancer Gene Ther 2008;15:126–32.CrossRefPubMedGoogle Scholar
  34. 34.
    Liu G, Mang’era K, Liu N, Gupta S, Rusckowski M, Hnatowich DJ. Tumor pretargeting in mice using (99m)Tc-labeled morpholino, a DNA analog. J Nucl Med 2002;43:384–91.PubMedGoogle Scholar
  35. 35.
    Liu G, He J, Dou S, Gupta S, Vanderheyden JL, Rusckowski M, et al. Pretargeting in tumored mice with radiolabeled morpholino oligomer showing low kidney uptake. Eur J Nucl Med Mol Imaging 2004;31:417–24.CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  1. 1.Division of Nuclear Medicine, Department of RadiologyUniversity of Massachusetts Medical SchoolWorcesterUSA
  2. 2.Yale PET Center, Department of Diagnostic RadiologyYale UniversityNew HavenUSA
  3. 3.Department of RadiologyUmass Medical SchoolWorcesterUSA

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