Trastuzumab-Doxorubicin Conjugate Provides Enhanced Anti-Cancer Potency and Reduced Cardiotoxicity


Since trastuzumab monotherapy for treatment of breast cancer with HER2/ErbB2 over-expression has been shown to have limited efficacy, combined treatment of trastuzumab with chemotherapy is widely practiced in clinic. However, certain combination treatments of trastuzumab and chemotherapy (i.e. doxorubicin) are not recommended due to high risk of cardiotoxicity. Antibody-drug conjugates (ADCs) offer selective delivery of cytotoxic agents into targeted cancer cells, thereby allowing for reduced general cellular cytotoxicity caused by chemotherapeutic agents through antibody mediated specific recognition of tumor antigens. In this study, we constructed a trastuzumab-doxorubicin conjugate (T-Dox) using a thioether linkage and characterized both biophysical stability and anti-cancer potency of the T-Dox using a panel of HER2 expressing cancer cell lines. The T-Dox conjugate showed significantly improved anti-cancer potency in comparison with trastuzumab. The results demonstrated for the first time that there were significant differences in the uptake of T-Dox among high HER2 expression cancer cells and higher T-Dox uptake also showed stronger anti-cancer potency. Similar to trastuzumab, T-Dox selectively bound to HER2 overexpressing cancer cells and low HER2 expression cells had no detectable uptake of T-Dox. Consistent to the uptake data, human cardiomyocyte cells had no detectable HER2 expression and T-Dox showed minimal cytotoxic effects. On the contrary, a treatment with combination of trastuzumab and doxorubicin showed severe cytotoxicity to human cardiomyocytes (>90% cell death after 3 day exposure). This study demonstrated that trastuzumab conjugated with doxorubicin (T-Dox) can provide valuable alternative to the combination treatment with doxorubicin and trastuzumab for high HER2 expressing cancer patients.

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N. Zhang, M. Klegerman, H. Deng, Y. Shi, E. Golunski and Z. An, "Trastuzumab-Doxorubicin Conjugate Provides Enhanced Anti-Cancer Potency and Reduced Cardiotoxicity," Journal of Cancer Therapy, Vol. 4 No. 1, 2013, pp. 308-322. doi: 10.4236/jct.2013.41038.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] L. Gianni, E. Salvatorelli and G. Minotti, “Anthracycline Cardiotoxicity in Breast Cancer Patients: Synergism with Trastuzumab and Taxanes,” Cardiovascular Toxicology, Vol. 7, No. 2, 2007, pp. 67-71. doi:10.1007/s12012-007-0013-5
[2] K. Y. Chan, P. Xiang, L. Zhou, K. Li, P. C. Ng, C. C. Wang, L. Zhang, H. Y. Deng, N. H. Pong, H. Zhao, W. Y. Chan and R. Y. Sung, “Thrombopoietin Protects against Doxorubicin-Induced Cardiomyopathy, Improves Cardiac Function, and Reversely Alters Specific Signalling Networks,” European Journal of Heart Failure, Vol. 13, No. 4, 2011, pp. 366-376. doi:10.1093/eurjhf/hfr001
[3] C. H. Chen, N. V. Cuong, Y. T. Chen, R. C. So, I. Liau and M. F. Hsieh, “Overcoming Multidrug Resistance of Breast Cancer Cells by the Micellar Doxorubicin Nano-particles of MPEG-PCL-Graft-Cellulose,” Journal of Nanoscience and Nanotechnology, Vol. 11, No. 1, 2011, pp. 53-60. doi:10.1166/jnn.2011.3102
[4] A. Gabizon, H. Shmeeda and Y. Barenholz, “Pharmacokinetics of Pegylated Liposomal Doxorubicin: Review of Animal and Human Studies,” Clinical Pharmacokinetics, Vol. 42, No. 5, 2003, pp. 419-436. doi:10.2165/00003088-200342050-00002
[5] H. D. King, A. J. Staab, K. Pham-Kaplita, D. Yurgaitis, R. A. Firestone, S. J. Lasch and P. A. Trail, “BR96 Conjugates of Highly Potent Anthracyclines,” Bioorganic & Medicinal Chemistry Letters, Vol. 13, No. 13, 2003, pp. 2119-2122. doi:10.1016/S0960-894X(03)00375-5
[6] S. Meschini, M. Condello, P. Lista, B. Vincenzi, A. Baldi, G. Citro, G. Arancia and E. P. Spugnini, “Electroporation Adopting Trains of Biphasic Pulses Enhances in Vitro and in Vivo the Cytotoxic Effect of Doxorubicin on Multidrug Resistant Colon Adenocarcinoma Cells (LoVo),” European Journal of Cancer, Vol. 48, No. 14, 2012, pp. 2236-2243. doi:10.1016/j.ejca.2011.11.031
[7] M. E. O’Brien, N. Wigler, M. Inbar, R. Rosso, E. Grischke, A. Santoro, R. Catane, D. G. Kieback, P. Tomczak, S. P. Ackland, F. Orlandi, L. Mellars, L. Alland and C. Tendler, “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,” Annals of Oncology, Vol. 15, No. 3, 2004, pp. 440-449. doi:10.1093/annonc/mdh097
[8] J. A. O’Shaughnessy, “Pegylated Liposomal Doxorubicin in the Treatment of Breast Cancer,” Clinical Breast Cancer, Vol. 4, No. 5, 2003, pp. 318-328. doi:10.3816/CBC.2003.n.037
[9] P. A. Trail, H. D. King and G. M. Dubowchik, “Monoclonal Antibody Drug Immunoconjugates for Targeted Treatment of Cancer,” Cancer Immunol Immunother, Vol. 52, No. 5, 2003, pp. 328-337.
[10] Y. Y. Diao, H. Y. Li, Y. H. Fu, M. Han, Y. L. Hu, H. L. Jiang, Y. Tsutsumi, Q. C. Wei, D. W. Chen and J. Q. Gao, “Doxorubicin-Loaded PEG-PCL Copolymer Micelles Enhance Cytotoxicity and Intracellular Accumulation of Doxorubicin in Adriamycin-Resistant Tumor Cells,” International Journal of Nanomedicine, Vol. 6, No. 9, 2011, pp. 1955-1962.
[11] B. Guo, H. L. Zhu, S. X. Li, X. C. Lu and H. Fan, “Individualized Liposomal Doxorubicin-Based Treatment in Elderly Patients with Non-Hodgkin’s Lymphoma,” Onkologie, Vol. 34, No. 4, 2011, pp. 184-188. doi:10.1159/000327008
[12] I. Kim, H. J. Byeon, T. H. Kim, E. S. Lee, K. T. Oh, B. S. Shin, K. C. Lee and Y. S. Youn, “Doxorubicin-Loaded Highly Porous Large PLGA Microparticles as a Sustained-Release Inhalation System for the Treatment of Metastatic Lung Cancer,” Biomaterials, Vol. 33, No. 2, 2012, pp. 5574-5583. doi:10.1016/j.biomaterials.2012.04.018
[13] X. W. Ma, Y. L. Zhao and X. J. Liang, “Nanodiamond Delivery Circumvents Tumor Resistance to Doxorubicin,” Acta Pharmacologica Sinica, Vol. 32, No. 5, 2011, pp. 543-544. doi:10.1038/aps.2011.58
[14] J. Niu, Z. Su, Y. Xiao, A. Huang, H. Li, X. Bao, S. Li, Y. Chen, M. Sun and Q. Ping, “Octreotide-Modified and PH-Triggering Polymeric Micelles Loaded with Doxorubicin for Tumor Targeting Delivery,” European Journal of Pharmaceutical Sciences, Vol. 45, No. 1-2, 2011, pp. 216-226. doi:10.1016/j.ejps.2011.11.013
[15] L. Zhao, L. Zhu, F. Liu, C. Liu, D. Shan, Q. Wang, C. Zhang, J. Li, J. Liu, X. Qu and Z. Yang, “PH Triggered Injectable Amphiphilic Hydrogel Containing Doxorubicin and Paclitaxel,” International Journal of Pharmaceutics, Vol. 410, No. 1-2, 2011, pp. 83-91. doi:10.1016/j.ijpharm.2011.03.034
[16] J. G. Reynolds, E. Geretti, B. S. Hendriks, H. Lee, S. C. Leonard, S. G. Klinz, C. O. Noble, P. B. Lucker, P. W. Zandstra, D. C. Drummond, K. J. Olivier, Jr., U. B. Nielsen, C. Niyikiza, S. V. Agresta and T. J. Wickham, “HER2-Targeted Liposomal Doxorubicin Displays Enhanced Anti-Tumorigenic Effects without Associated Cardiotoxicity,” Toxicology and Applied Pharmacology, Vol. 262, No. 1, 2012, pp. 1-10. doi:10.1016/j.taap.2012.04.008
[17] A. G. Garcia, H. Nedev, K. Bijian, J. Su, M. A. Alaoui-Jamali and H. U. Saragovi, “Reduced in Vivo Lung Metastasis of a Breast Cancer Cell Line after Treatment with Herceptin MAB Conjugated to Chemotherapeutic Drugs,” Oncogene, Vol. 283, No. 7, 2012, pp. 1-7.
[18] E. L. Sievers and P. D. Senter, “Antibody-Drug Conjugates in Cancer Therapy,” Annual Review of Medicine, Vol. 64, No. 10, 2012, pp. 15-29.
[19] M. Y. Yeh, S. R. Roffler and M. H. Yu, “Doxorubicin: Monoclonal Antibody Conjugate for Therapy of Human Cervical Carcinoma,” International Journal of Cancer, Vol. 51, No. 2, 1992, pp. 274-282. doi:10.1002/ijc.2910510217
[20] H. A. Burris, 3rd, J. Tibbitts, S. N. Holden, M. X. Sliwkowski and G. D. Lewis Phillips, “Trastuzumab Emtansine (T-DM1): A Novel Agent for Targeting HER2+ Breast Cancer,” Clinical Breast Cancer, Vol. 11, No. 5, 2011, pp. 275-282. doi:10.1016/j.clbc.2011.03.018
[21] D. J. Slamon, B. Leyland-Jones, S. Shak, H. Fuchs, V. Paton, A. Bajamonde, T. Fleming, W. Eiermann, J. Wolter, M. Pegram, J. Baselga and L. Norton, “Use of Chemotherapy plus a Monoclonal Antibody against HER2 for Metastatic Breast Cancer That Overexpresses HER2,” The New England Journal of Medicine, Vol. 344, No. 11, 2001, pp. 783-792. doi:10.1056/NEJM200103153441101
[22] G. L. Griffiths, M. J. Mattes, R. Stein, S. V. Govindan, I. D. Horak, H. J. Hansen and D. M. Goldenberg, “Cure of SCID Mice Bearing Human B-Lymphoma Xenografts by an Anti-CD74 Antibody-Anthracycline Drug Conjugate,” Clinical Cancer Research, Vol. 9, No. 17, 2003, pp. 6567-6571.
[23] C. D. Austin, A. M. De Maziere, P. I. Pisacane, S. M. van Dijk, C. Eigenbrot, M. X. Sliwkowski, J. Klumperman and R. H. Scheller, “Endocytosis and Sorting of ErbB2 and the Site of Action of Cancer Therapeutics Trastuzumab and Geldanamycin,” Molecular Biology of the Cell, Vol. 15, No. 12, 2004, pp. 5268-5282. doi:10.1091/mbc.E04-07-0591
[24] H. K. Erickson, P. U. Park, W. C. Widdison, Y. V. Kovtun, L. M. Garrett, K. Hoffman, R. J. Lutz, V. S. Goldmacher and W. A. Blattler, “Antibody-Maytansinoid Conjugates Are Activated in Targeted Cancer Cells by Lysosomal Degradation and Linker-Dependent Intracellular Processing,” Cancer Research, Vol. 66, No. 8, 2006, pp. 4426-4433. doi:10.1158/0008-5472.CAN-05-4489
[25] S. Ha, Y. Ou, J. Vlasak, Y. Li, S. Wang, K. Vo, Y. Du, A. Mach, Y. Fang and N. Zhang, “Isolation and Characterization of IgG1 with Asymmetrical Fc Glycosylation,” Glycobiology, Vol. 21, No. 8, 2011, pp. 1087-1096. doi:10.1093/glycob/cwr047
[26] X. Fan, R. J. Brezski, M. Fa, H. Deng, A. Oberholtzer, A. Gonzalez, W. P. Dubinsky, W. R. Strohl, R. E. Jordan, N. Zhang and Z. An, “A Single Proteolytic Cleavage within the Lower Hinge of Trastuzumab Reduces Immune Effector Function and in Vivo Efficacy,” Breast Cancer Research, Vol. 14, No. 4, 2012, p. R116. doi:10.1186/bcr3240
[27] M. E. Klegerman, A. J. Hamilton, S. L. Huang, S. D. Tiukinhoy, A. A. Khan, R. C. MacDonald and D. D. Mc-Pherson, “Quantitative Immunoblot Assay for Assessment of Liposomal Antibody Conjugation Efficiency,” Analytical Biochemistry, Vol. 300, No. 1, 2002, pp. 46-52. doi:10.1006/abio.2001.5443
[28] N. Zhang, L. Liu, C. D. Dumitru, N. R. Cummings, M. Cukan, Y. Jiang, Y. Li, F. Li, T. Mitchell, M. R. Mallem, Y. Ou, R. N. Patel, K. Vo, H. Wang, I. Burnina, B. K. Choi, H. E. Huber, T. A. Stadheim and D. Zha, “Glycoengineered Pichia Produced Anti-HER2 is Comparable to Trastuzumab in Preclinical Study,” Mabs, Vol. 3, No. 3, 2011, pp. 289-298.
[29] G. D. Lewis Phillips, G. Li, D. L. Dugger, L. M. Crocker, K. L. Parsons, E. Mai, W. A. Blattler, J. M. Lambert, R. V. Chari, R. J. Lutz, W. L. Wong, F. S. Jacobson, H. Koeppen, R. H. Schwall, S. R. Kenkare-Mitra, S. D. Spencer and M. X. Sliwkowski, “Targeting HER2-Positive Breast Cancer with Trastuzumab-DM1, an Antibody-Cytotoxic Drug Conjugate,” Cancer Research, Vol. 68, No. 22, 2008, pp. 9280-9290. doi:10.1158/0008-5472.CAN-08-1776
[30] N. L. Spector and K. L. Blackwell, “Understanding the Mechanisms Behind Trastuzumab Therapy for Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer,” Journal of Clinical Oncology, Vol. 27, No. 34, 2009, pp. 5838-5847. doi:10.1200/JCO.2009.22.1507
[31] M. X. Sliwkowski, J. A. Lofgren, G. D. Lewis, T. E. Hotaling, B. M. Fendly and J. A. Fox, “Nonclinical Studies Addressing the Mechanism of Action of Trastuzumab (Herceptin),” Seminars in Oncology, Vol. 26, No. 4, 1999, pp. 60-70.
[32] P. M. LoRusso, D. Weiss, E. Guardino, S. Girish and M. X. Sliwkowski, “Trastuzumab Emtansine: A Unique Antibody-Drug Conjugate in Development for Human Epidermal Growth Factor Receptor 2-Positive Cancer,” Clinical Cancer Research, Vol. 17, No. 20, 2011, pp. 6437-6447. doi:10.1158/1078-0432.CCR-11-0762
[33] R. Mandler, H. Kobayashi, E.R. Hinson, M. W. Brechbiel and T. A. Waldmann, “Herceptin-Geldanamycin Immunoconjugates: Pharmacokinetics, Biodistribution, and Enhanced Antitumor Activity,” Cancer Research, Vol. 64, No. 9, 2004, pp. 1460-1467. doi:10.1158/0008-5472.CAN-03-2485
[34] C. Marx, J. M. Held, B. W. Gibson and C. C. Benz, “ErbB2 Trafficking and Degradation Associated with K48 and K63 Polyubiquitination,” Cancer Research, Vol. 70, No. 22, 2010, pp. 3709-3717. doi:10.1158/0008-5472.CAN-09-3768
[35] S. M. Raja, R. J. Clubb, M. Bhattacharyya, M. Dimri, H. Cheng, W. Pan, C. Ortega-Cava, A. Lakku-Reddi, M. Naramura, V. Band and H. Band, “A Combination of Trastuzumab and 17-AAG induces enhanced ubiquitinylation and lysosomal pathway-dependent ErbB2 degradation and cytotoxicity in ErbB2-overexpressing breast cancer cells,” Cancer Biology & Therapy, Vol. 7, No. 10, 2008, pp. 1630-1640. doi:10.4161/cbt.7.10.6585

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