Herceptin Conjugates Linked by EDC Boost Direct Tumor Cell Death via Programmed Tumor Cell Necrosis Jiemiao Hu1., Xinli Liu2., Dennis Hughes1, Francisco J. Esteva3, Bolin Liu4, Joya Chandra1, Shulin Li1* 1 Department of Pediatric Research, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America, 2 Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas, United States of America, 3 Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America, 4 Department of Pathology, University of Colorado Denver School of Medicine, Aurora, Colorado, United States of America Abstract Tumor-targeted antibody therapy is one of the safest biological therapeutics for cancer patients, but it is often ineffective at inducing direct tumor cell death and is ineffective against resistant tumor cells. Currently, the antitumor efficacy of antibody therapy is primarily achieved by inducing indirect tumor cell death, such as antibody-dependent cell cytotoxicity. Our study reveals that Herceptin conjugates, if generated via the crosslinker EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), are capable of engendering human epidermal growth factor receptor 2 (Her2) positive tumor cells death. Using a high-performance liquid chromatography (HPLC) system, three peaks with estimated molecular weights of antibody monomer, dimer, and trimer were isolated. Both Herceptin trimer and dimer separated by HPLC induced significant levels of necrotic tumor cell death, although the trimer was more effective than the dimer. Notably, the Herceptin trimer also induced Herceptin-resistant tumor cell death. Surprisingly different from the known cell death mechanism that often results from antibody treatment, the Herceptin trimer elicited effective and direct tumor cell death via a novel mechanism: programmed cell necrosis. In Her2-positive cells, inhibition of necrosis pathways significantly reversed Herceptin trimer- induced cell death. In summary, the Herceptin trimer reported herein harbors great potential for overcoming tumor cell resistance to Herceptin treatment. Citation: Hu J, Liu X, Hughes D, Esteva FJ, Liu B, et al. (2011) Herceptin Conjugates Linked by EDC Boost Direct Tumor Cell Death via Programmed Tumor Cell Necrosis. PLoS ONE 6(8): e23270. doi:10.1371/journal.pone.0023270 Editor: Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America Received May 16, 2011; Accepted July 10, 2011; Published August 10, 2011 Copyright: 2011 Hu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the National Institutes of Health (grant R01CA120895-3S). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sli4@mdanderson.org . These authors contributed equally to this work. Introduction Human epidermal growth factor receptor 2 (Her2) is overex- pressed in many types of cancers. [1,2]. Herceptin, also known as Trastuzumab, is a humanized recombinant monoclonal antibody that binds to the extracellular domain of Her2 and is the first Her2 antibody approved by the US Food and Drug Administration for treating cancers in humans. Antibody therapy provides excellent tumor specificity; however, the clinical response to Herceptin therapy has not been very strong, with only 12–34% tumor remission noted over 9 months in metastatic breast cancer patients in early clinical trials [3]. Moreover, both primary resistance and acquired resistance to Herceptin were observed, thus limiting broad application of this safe therapy [4]. Further improvement of Herceptin’s therapeutic effect is needed. Unlike chemotherapy, Herceptin does not directly cause tumor cell death. Like many other targeted antibodies, Herceptin induces Her2-positive tumor cell death via ADCC [5,6,7]. In the current study, we sought to test whether Herceptin conjugate promotes induction of direct tumor cell death and whether such effect may also overcome tumor resistance to antibody treatment. We discovered that, regardless of tumor cell resistance to wild- type Herceptin, the Herceptin conjugate generated using EDC but not the crosslinker SMCC (succinimidyl-4-(N-maleimidomethyl)- cyclohexane-1-carboxylate)as found by others [8], boosted direct tumor cell death via inducing programmed tumor cell necrosis. The Herceptin trimer conjugate was more effective than the Herceptin dimer conjugate in inducing Her2-positive tumor cell death. The Herceptin conjugate did not cause death of any Her2- negative or underexpressing tumor cells, illustrating this conju- gate’s specificity and potential safety as a therapeutic agent. To our knowledge, our report is the first to reveal the capability of a tumor-targeted antibody to simultaneously induce programmed necrotic tumor cell death (PNCD) and overcome the resistance of tumor cells to antibody treatment. Results Generation of Herceptin conjugates The anti-Her2 antibody, Herceptin, has proven effective in blocking the Her2 downstream signaling pathway [9,10,11] and in sensitizing Her2-expressing tumor cells to other treatments [12,13]; however, there is no evidence that Herceptin alone induces potent tumor cell death. The hypothetical basis of this study tested whether the oligomerization of Herceptin immuno- globulin G (IgG) causes direct and effective tumor cell death. The rationale for generating this hypothesis preceded from the fact that immunoglobulin M (IgM), an immunoglobulin pentamer, causes PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23270