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An anti-transferrin receptor antibody enhanced the growth inhibitory effects of chemotherapeutic drugs on human glioma cells
International Immunopharmacology 11 (2011) 1844–1849
Contents lists available at ScienceDirect
International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i n t i m p
An anti-transferrin receptor antibody enhanced the growth inhibitory effects of chemotherapeutic drugs on human glioma cells☆ Guozheng Xu a, 1, Xue Wen a,⁎, 1, Yi Hong b, c, Hao Du a, Xinyuan Zhang a, Jian Song a, Yimei Yin a, He Huang a, Guanxin Shen b a b c
Department of Neurosurgery, Wuhan General Hospital of Guangzhou Command, Wuhan 430070, China Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China Department of Pharmacy, Hubei University of Chinese Medicine, Wuhan 430065, China
a r t i c l e
i n f o
Article history: Received 31 March 2011 Received in revised form 18 July 2011 Accepted 18 July 2011 Available online 3 August 2011 Keywords: Glioma Transferrin receptor Monoclonal antibody Nimustine Chemoimmunotherapy
a b s t r a c t Transferrin receptor (TfR) has been used as a target for antibody-based therapy of cancer. Anti-TfR antibody together with chemotherapeutic drugs has potential for cancer therapy. In this study, we investigated the in vitro anti-tumor effects of the anti-TfR monoclonal antibody (mAb), 7579, alone or in combination with Nimustine, a chemotherapeutic drug, on the gliomas cell lines U251 and U87MG. Our results indicated that 7579 alone dramatically down-regulated surface expression of TfR on tumor cells and induced S phase accumulation and apoptosis of tumor cells. Compared with 7579 or Nimustine used alone, the combination of 7579 with Nimustine demonstrated enhanced growth inhibitory effect on tumor cells. PI (Propidium iodide)/ Annexin V staining analyzed by FCM (flow cytometry) demonstrated that 7579 enhanced the cytotoxic effects of chemotherapeutic drug on tumor cells, indicating the therapeutic effect of 7579 was mediated mainly by promoting tumor cell necrosis. Using the median-effect/combination-index isobologram method, we further evaluated the nature of 7579/chemotherapeutic drug interactions. Synergistic interaction was observed for combination of 7579 with Nimustine. Our study provides additional evidence to develop combination therapies of anti-TfR mAbs-plus chemoimmunotherapy for gliomas. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Gliomas are the most frequent primary central nervous system (CNS) neoplasms, accounting for more than 50% of all brain tumors [1]. Despite advances in diagnostic and surgical techniques, the prognosis of these patients remains poor and the median survival of 12 months has not significantly changed during the last several years [2]. The latter is primarily due to the fact that gliomas are highly resistant to chemotherapy and radiotherapy [3]. Transferrin receptor (TfR, CD71) plays a crucial role in the cellular uptake of iron and in the regulation of cell growth [4]. In malignant tissues, TfR is expressed more abundantly than in normal tissues [5]. Therefore, TfR could be a relevant target for antibody-based therapies against tumors [6–8]. Due to the in vitro and in vivo success of anti-TfR antibody in tumor cell killing [9–11], a Phase I clinical trial of an IgA monoclonal anti-Tf receptor antibody 42/6 was conducted in 27 patients with advanced refractory cancer of different origins [12]. The
☆ There is no conflict of interest in our research. ⁎ Corresponding author. Tel.: + 86 2768878528; fax: + 86 2787212690. E-mail address: [email protected] (X. Wen). 1 These authors contributed equally and share the first author. 1567-5769/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2011.07.014
treatment was well tolerated, highlighting the safety of anti-TfR immunotherapy. In vitro combination therapy of an anti-TfR MAb, A24, and Zidovudine demonstrated improved cytotoxic effects on adult T-cell leukemia cells [13]. In vitro, A24 blocked proliferation of mantle cell lymphoma cells (MCL) by inducing mantle cell lymphoma cell apoptosis through caspase-3 and caspase-9 activation, either alone or synergistically with chemotherapeutic agents Adriblastin,Ara-C and Etoposide [14]. MAb A24 prevented tumor establishment and decreased the growth rate of pre-established MCL tumors in athymic nude mice. Therefore, A24-based therapies alone or in association with classic chemotherapies could provide a new alternative strategy against MCL, particularly in relapsing cases. In general, hematopoietic tumors are particularly suitable for treatment using TfR-targeting therapeutics since they express high levels of TfR and are more sensitive to the inhibitory effect of anti-TfR antibodies than other malignancies [15]. Nevertheless, elevated levels of TfR expression are widely observed on non-hematopoietic tumor cells and correlated with tumor grade and stage or poor prognosis [16–19]. Thus, TfR is also considered as an attractive target for nonhematopoietic tumor therapy. Trowbridge et al. reported that an antiTfR antibody could be efficient in inhibiting human melanoma xenograft growth in athymic mice [7]. In addition, combinations of two anti-TfR mAbs had anti-proliferative effects on breast cancer cells in vitro and in vivo [4]. To explore the combination use of anti-TfR
G. Xu et al. / International Immunopharmacology 11 (2011) 1844–1849
mAbs with chemotherapeutic drugs for cancer chemoimmunotherapy may be a promising strategy for glioma chemoimmunotherapy. Alkylating drugs are the oldest class of anti-cancer drugs which are still commonly in use, and they remain important in the treatment of several types of cancers. Nimustine (ACNU) is a chloroethylating agent which has been used either alone or in combination with other agents for the treatment of glioma [20]. 7579 is a murine monoclonal anti-human TfR IgG antibody made in our lab. It is a potent growth inhibitor of the erythroleukemia cell line K562 [21]. In this study, we investigated the in vitro effects of 7579 and Nimustine individually or in combination with each other on TfR overexpressing glioma cell lines and fresh glioma tissue, including the effects on apotosis, cell cycle arrest, TfR expression, and cell proliferation. The cell lines used were U251 which is a human glioblastoma cell line and U87MG which is a human malignant glioma cell line.
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1.1. Materials and methods 1.1.1. Cells, antibodies and chemotherapeutic drugs The glioma cell lines U251 and U87MG cells were purchased from the Shanghai Institute for Biological Science of the Chinese Academy of Science. Cells were maintained in DMEM supplemented with 10% fetal bovine serum and incubated at 37 °C in a humidified atmosphere containing 5% CO2. 7579, an anti-TfR MAb, was purified and characterized in our laboratory as described previously [21]. Antibody to β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-CD71 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Non-specific mouse IgG and horseradish peroxidase (HRP)-conjugated goat antimouse IgG antibodies were from Southern Biotech (Birmingham, USA). Chemotherapeutic Nimustine was purchased from Daiichi Sankyo (Japan).
Fig. 1. Transferrin receptor (TfR) expression in glioma tissues and glioma cells. (A) TfR staining in the tissues glioma tissues (b: grade II; c: grade III; d: grade IV) and normal brain tissues. (B) TfR staining in U251 and U87MG cell lines. (C) Western blot analysis of TfR expression in the tissue of glioma and normal brain. Data are presented as means ± SEM from three separate experiments. The average signal intensity was standardized to β-actin. The ratio of TfR expression to β-actin expression was shown. (D) Western blot analysis of TfR expression in U251 and U87MG cell lines. The average signal intensity was standardized to β-actin. The ratio of TfR expression to β-actin expression was shown. Data were shown as means ± SD of triplicate experiments.*p b 0.01.
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1.1.2. Sample and clinical information Normal tissues (n= 3) and brain tumors (n= 20) were obtained from glioma patients undergoing primary resection or biopsy of their tumor at the department of Neurosurgery, Wuhan General Hospital of Guangzhou Command (Wuhan, China). There are three grades: lowgrade astrocytomas (LGA; gradeII) (n= 8); anaplastic oligodendrogliomas (grade III) (n= 7); and glioblastoma multiforme (GBM; gradeIV) (n= 5). Collection of the human tissue specimens was approved by the appropriate ethics committee and was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. All persons gave informed consent prior to their inclusion in the study. 1.1.3. Immunohistochemical staining and evaluation Immunohistochemical analysis of TfR expression in tumor tissues and cell lines was performed with mouse anti-CD71 antibody as the primary antibody, and with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody as the secondary antibody. 1.1.4. Western blot analysis Western blot analysis of TfR expression in tumor tissues and cells was performed with mouse anti-CD71 antibody as primary antibody, and followed by horseradish peroxidase (HRP)-conjugated goat antimouse IgG antibody as the secondary antibody. The protein band density was quantified using the ImageJ software (Version 1.38x),
with β-actin as a loading control. The relative expression of TfR protein was expressed as the ratio of TfR expression to β-actin expression. 1.1.5. Cell apoptosis analysis U251 and U87MG cells (2 × 105) were placed in 6-well plates and treated with 7579 (100 μg/mL) alone, chemotherapeutic drugs (Nimustine) (40 μg/mL and 60 μg/mL) alone or the combinations of 7579 and Nimustine for 72 h. The same concentration of nonspecific mouse IgG (100 μg/mL) was used for isotype controls. Tumor cells were then washed with ice-cold PBS and incubated with a combination of 10 μg/mL FITC–Annexin V and 50 μg/mL PI (Roche Diagnostics, Mannheim, Germany) [22] at room temperature for 30 min. Analysis was performed by FCM (Becton–Dickinson LSR II). 1.1.6. Cell proliferation analysis U251 and U87MG cells (2 × 105) were grown in 6-well plates and treated with 7579 alone (100 μg/mL), Nimustine alone (U251: 40 μg/mL, U87MG: 60 μg/mL) or a combination of 7579 and Nimustine. The same concentration of nonspecific mouse IgG was used for isotype controls. Tumor cells were labeled with carboxyfluorescein diacetate (CFSE) at time zero and then grown for 72 hours with the agents noted above. The cellular concentration of CFSE is reduced by 50% with each cycle with a resultant change in flourscent color. After 72 h of culture tumor cells were washed with ice-cold PBS and analysed by flow
Fig. 2. Combined effects of 7579 and Nimustine on apoptosis of glioma cells. Apoptosis of U251 cells detected with FCM after treated with the 7579 and Nimustine as indicated, alone or in combination, for 72 h. (B) Apoptosis of U87MG cells detected with FCM after treated with the 7579 and Nimustine as indicated, alone or in combination, for 72 h. (C)The statistical analysis of apoptosis of U251 cells. (D)The statistical analysis of apoptosis of U87MG cells. Data represent the means and standard deviation of 4 independent experiments. *p b 0.01.
G. Xu et al. / International Immunopharmacology 11 (2011) 1844–1849
cytometry. The data from 10,000 cells were collected and analyzed using ModiFit Software. 1.1.7. Cell cycle analysis U251 and U87MG cells (2× 105) were grown in 6-well plates and treated with 7579 alone (100 μg/mL), Nimustine alone (U251: 40 μg/mL, U87MG: 60 μg/mL) or the combinations of 7579 and Nimustine for 72 h. The same concentration of nonspecific mouse IgG was used for isotype controls. Tumor cells were harvested and fixed with 70% ethanol at 4 °C overnight, and then stained with 50 μg/mL PI for 30 min. Analysis was performed by FCM. The data from 10,000 cells were collected and analyzed using ModiFit Software. 1.1.8. Statistical analysis Statistical significance of the data was calculated by ANVOA or Student's t-test. A significance level of p b 0.01was chosen. Both analyses were carried out using SPSS 10.0 statistical software. 1.2. Results 1.2.1. TfR expression in glioma specimens and cell lines We examined the TfR expression in glioma specimens and normal cells by immunohistochemistry and western blot. We tested different types of glial tumors using tumor samples from LGA (grade II), anaplastic oligodendrogliomas (grade III), and GBMs (grade IV). The expression of TfR in these tumors was compared with that of normal cells from patients undergoing resection or biopsy. We found that TfR
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expression was up-regulated in glioma specimens and that the expression level was positively correlated with tumor grade (Fig. 1A). The glioma cell lines U251 and U87MG expressed high levels of TfR. 1.2.2. Apoptosis effects of 7579 and chemotherapeutic drug combination on glioma cells The apoptosis of cells after treatment with a combination of 7579 and Nimustine or each agent alone for 72 h was evaluated by FCM with PI-Annexin V staining. As shown in Fig. 2, both the combination of 7579 and Nimustine and each agent alone induced the death of tumor cells (U251: 53, 37, and 9%, (*p b 0.01), respectively and for U87MG: 47, 31 and 8%, (*p b 0.01), respectively). Thus, compared with Nimustine or 7579 when used alone, combination treatment with 7579 and Nimustine enhanced apoptosis of tumor cells and appeared to be slightly more than additive. 1.2.3. Proliferation inhibitory effects of 7579 and chemotherapeutic drug combinations with on glioma cells The proliferation of glioma cells after treatment with the combination of 7579 and Nimustine or each agent alone for 72 h was evaluated by FCM with CFSE staining. As shown in Fig. 3, the combination and each agent alone inhibited proliferation of tumor cells (U251: 5.4, 7.9, and 11.2, (*p b 0.01), respectively and for U87MG: 5.1, 7.6 and 8.4, (*p b 0.01), respectively). Thus, compared with Nimustine or 7579 alone, treatment with the combination of 7579 and Nimustine exhibited increased inhibition of proliferation of tumor cells.
Fig. 3. Combined effects of 7579 and Nimustine on proliferation of glioma cells. (A) Proliferation of U251 cells detected with FCM after treated with the 7579 and Nimustine as indicated, alone or in combination, for 72 h. (B) Proliferation of U87MG cells detected with FCM after treated with the 7579 and Nimustine as indicated, alone or in combination, for 72 h. (C) The statistical analysis of proliferation of U251 cells. (D) The statistical analysis of proliferation of U87MG cells. Data represent the means and standard deviation of 4 independent experiments. *p b 0.01.
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Fig. 4. Cell cycle phase distribution of glioma cells treated with the 7579 and Nimustine alone or in combination. (A) U251 cells were treated with 7579/Nimustine combinations or each agent alone for 72 h. DNA contents were measured by PI staining. (B) U87MG cells were treated with 7579/Nimustine combinations or each agent alone for 72 h. DNA contents were measured by PI staining. Histograms present one out of 4 independent experiments. (C) Cell cycle phase distribution of U251 cells treated with 7579/Nimustine combinations or each agent alone. (D) Cell cycle phase distribution of U87MG cells treated with 7579/Nimustine combinations or each agent alone. Data represent the means and standard deviation of 4 independent experiments. *p b 0.01.
1.2.4. Effects of 7579 and chemotherapeutic drug combination on the cell cycle of glioma cells Fig. 4 showed the cell cycle distribution of tumor cells after treatment with the combination of 7579 and Nimustine, or each agent alone for 72 h as evaluated by FCM with PI staining. Complete results are given in Fig. 4. Briefly, both agents alone decreased the number of cells in G1 and the combination provided and a further decrease that was essentially additive (12%, 19%, and 28%, respectively for U251 cells) with similar results on the U87MG cell line that appeared significantly more than additive for the combination (12%, 28%, and 54%, respectively). Cells in S phase were increased with cells in G2 showed inconsistent changes. 2. Discussion Most normal cells other than immature erythroid cells are low in transferrin receptor (TfR) expression. However, actively proliferating tumor cells exhibit high levels of TfR [23]. The growth inhibitory property of anti-TfR antibodies has been appreciated since the 1980s [24]. As for the mechanisms of anti-TfR antibody mediated growth inhibition, different antibodies have shown different modes of action in different models [25,26]. We have previously reported the construction of an intracellular antibody anti-TfR scFv-intrabody [5] and a human–mouse chimeric antibody D2C [21], which displayed a tumor-specifc distribution and had a strong antitumor effect. In this study, we showed that glioma specimens as well as cell lines exhibited abundant expression of TfR, and that expression of TfR correlated positively with tumor grade. These results suggested that TfR is a promising target for therapeutic strategy in gliomas. We showed that a murine monoclonal anti-human TfR IgG antibody 7579 induced S phase accumulation and apoptosis in 7579treated tumor cells with a resultant anti-proliferative effect. Further, combination treatment with 7579 and Nimustine was more effective
than either agent alone. These biological effects provide a basis for mAb/chemotherapeutic drug combination therapy, and also help explain the underlying mechanisms of combined efficacy. The blood brain barrier (BBB) is a bidirectional transport system [27] and enables the receptor-mediated transcytosis of either transferrin (Tf) or a TfR mAb from the blood to the brain [28]. The transferrin receptor (TfR) is expressed at both the BBB and the brain cell membrane (BCM) [29], and prior work in rats showed that the murine OX26 mAb against the rat TfR could deliver a PNA radiopharmaceutical to experimental brain tumors in vivo [30]. In the study by Lee et al., the 8D3 rat mAb to the mouse TfR [31] is used as a brain drug-targeting vector to enable imaging of gene expression in vivo in a transgenic mouse model of Huntington's disease (HD). Because our anti-TfR mAb (7579) is a murine monoclonal anti-human TfR IgG antibody it cannot permeate through the BBB of a mouse, so it cannot be tested in a mouse model. In conclusion, we analyzed the in vitro therapeutic effects of 7579 and Nimustine alone and in combination with each other on glioma cells. The results suggest that combination therapy was more effective than the use of either agent alone. Our study helps clarify the mechanism of action of TfR-directed therapies, suggesting the addition of this targeted approach to currently available cancer treatments might be a better therapeutic strategy. We declare that we have no conflict of interest. Acknowledgements This work was supported by Grant from the Hospital foundation of Wuhan General Hospital of Guangzhou Command (NO. 201010). References [1] Kleihues P, Louis DN, Scheithauer BW, Rorke LB, Reifenberger G, Burger PC, et al. The WHO classification of tumors of the nervous system. J Neuropathol Exp Neurol 2002;61:215–25.
G. Xu et al. / International Immunopharmacology 11 (2011) 1844–1849 [2] Brandes AA. State-of-the-art treatment of high-grade brain tumors. Semin Oncol 2003;30:4–9. [3] Prados MD, Levin V. Biology and treatment of malignant glioma. Semin Oncol 2000;27:1–10. [4] Jone DT, Trowbridge IS, Harris AL. Effects of transferrin receptor blockade on cancer cell proliferation and hypoxia-inducible factor function and their differential regulation by ascorbate. Cancer Res 2006;66:2749–56. [5] Peng JL, Wu S, Zhao XP, Wang M, Li WH, Shen X, et al. Downregulation of transferrin receptor surface expression by intracellular antibody. Biochem Biophys Res Commun 2007;354:864–71. [6] Ng PP, Dela Cruz JS, Sorour DN, Stinebaugh JM, Shin SU, Shin DS, et al. An antitransferrin receptor-avidin fusion protein exhibits both strong proapoptotic activity and the ability to deliver various molecules into cancer cells. Proc Natl Acad Sci USA 2002;99:10706–11. [7] Trowbridge IS, Domingo DL. Anti-transferrin receptor monoclonal antibody and toxinantibody conjugates affect growth of human tumor cells. Nature 1981;294: 171–3. [8] Domingo DL, Trowbridge IS. Transferrin receptor as a target for antibody-drug conjugates. Methods Enzymol 1985;112:238–47. [9] Okamoto K, Harada K, Ikeyama S, Iwasa S. Therapeutic effect of ansamitocin targeted to tumor by a bispecific monoclonal antibody. Jpn J Cancer Res 1992;83 (7):761–8. [10] Graziadei I, Gaggl S, Kaserbacher R, Braunsteiner H, Vogel W. The acute-phase protein alpha 1-antitrypsin inhibits growth and proliferation of human early erythroid progenitor cells (burst-forming units-erythroid) and of human erythroleukemic cells (K562) in vitro by interfering with transferrin iron uptake. Blood 1994;83(1):260–8. [11] Chan SM, Hoffer PB, Maric N, Duray P. Inhibition of gallium-67 uptake in melanoma by an anti-human transferrin receptor monoclonal antibody. J Nucl Med 1987;28(8):1303–7. [12] Brooks D, Taylor C, Dos Santos B, Linden H, Houghton A, Hecht TT, et al. Phase Ia trial of murine immunoglobulin an antitransferrin receptor antibody 42/6. Clin Cancer Res 1995;11:1259–65. [13] Moura IC, Lepelletier Y, Arnulf B, England P, Baude C, Beaumont C, et al. A neutralizing monoclonal antibody (mAb/A24) directed against the transferrin receptor induces apoptosis of tumor T lymphocytes from ATL patients. Blood 2004;103:1838–45. [14] Lepelletier Y, Camara-Clayette V, Jin H, Hermant A, Coulon S, Dussiot M, et al. Prevention of mantle lymphoma tumor establishment by routing transferrin receptor toward lysosomal compartments. Cancer Res 2007;67:1145–54. [15] Ng PP, Helguera G, Daniels TR, Lomas SZ, Rodriguez JA, Schiller G, et al. Molecularevents contributing to cell death in malignant human hematopoietic cells elicited by an IgG3-avidin fusion protein targeting the transferrin receptor. Blood 2006;108:2745–54. [16] Kondo K, Noguchi M, Mukai K, Matsuno Y, Sato Y, Shimosato Y, et al. Transferrin receptor expression in adenocarcinoma of the lung as a histopathologic indicator of prognosis. Chest 1990;97:1367–71.
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[17] Seymour GJ, Walsh MD, Lavin MF, Strutton G, Gardiner RA. Transferrin receptor expression by human bladder transitional cell carcinomas. Urol Res 1987;15: 341–4. [18] Yang DC, Wang F, Elliott RL, Head JF. Expression of transferrin receptor and ferritin H-chain mRNA are associated with clinical and histopathological prognostic indicators in breast cancer. Anti-cancer Res 2001;21:541–9. [19] Ryschich E, Huszty G, Knaebel HP, Hartel M, Buchler MW, Schmidt J. Transferrin receptor is a marker of malignant phenotype in human pancreatic cancer and in neuroendocrine carcinoma of the pancreas. Eur J Cancer 2004;40:1418–22. [20] Gwak HS, Youn SM, Kwon AH, Lee SH, Kim JH, Rhee CH. ACNU-cisplatin continuous infusion chemotherapy as salvage therapy for recurrent glioblastomas: phase II study. J Neurooncol 2005;75:173–80. [21] Qing Y, Shuo W, Zhihua W, Huifen Z, Ping L, Lijiang L, et al. The in vitro antitumor effect and in vivo tumor-specificity distribution of human–mouse chimeric antibody against transferrin receptor. Cancer Immunol Immunother 2006;55:1111–21. [22] van Heerde Waander L, Robert-Offerman Saskia, Dumont Ewald, Hofstra Leo, Doevendans Pieter A, Smits Jos FM, et al. Markers of apoptosis in cardiovascular tissues: focus on Annexin V. Cardiovasc Res 2000;5:549–59. [23] Cheng Y, Zak O, Aisen P, Harrison SC, Walz T. Structure of the human transferrin receptor-transferrin complex. Cell 2004;116:565–76. [24] Kemp JD, Thorson JA, Mcalmont TH, Horowitz M, Cowdery JS, Ballas ZK. Role of the transferring receptor in lymphocyte growth: a rat IgG monoclonal antibody against the murine transferrin receptor produces highly selective inhibition of T and B cell activation protocols. J Immunol 1987;138:2422–6. [25] Trowbridge IS, Lopez F. Monoclonal antibody to transferrin receptor blocks transferrin binding and inhibits human tumor cells growth in vitro. Proc Natl Acad Sci USA 1982;79:1175–9. [26] Lesley JF, Schulte RJ. Inhibition of cell growth by monoclonal anti-transferrin receptor antibodies. Mol Cell Biol 1985;5:1814–21. [27] Zhang Y, Pardridge WM. Rapid transferrin efflux from brain to blood across the blood–brain barrier. J Neurochem 2001;76:1597–600. [28] Skarlatos S, Yoshikawa T, Pardridge WM. Transport of [125I] transferrin through the rat blood–brain barrier in vivo. Brain Res 1995;683:164–71. [29] Lee Hwa Jeong, Boado Ruben J, Braasch Dwaine A, Corey David R, William M, Pardridge MD. Imaging gene expression in the brain in vivo in a transgenic mouse model of Huntington's disease with an antisense radiopharmaceutical and drugtargeting technology. J Nucl Med 2002;43:948–56. [30] Shi N, Boado RJ, Pardridge WM. Antisense imaging of gene expression in the brain in vivo. Proc Natl Acad Sci USA 2000;97:14709–14. [31] Lee HJ, Engelhardt B, Lesley J, Bickel U, Pardridge WM. Targeting rat antimouse transferrin receptor monoclonal antibodies through the blood–brain barrier. J Pharmacol Exp Ther 2000;292:1048–52.
Contents lists available at ScienceDirect
International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i n t i m p
An anti-transferrin receptor antibody enhanced the growth inhibitory effects of chemotherapeutic drugs on human glioma cells☆ Guozheng Xu a, 1, Xue Wen a,⁎, 1, Yi Hong b, c, Hao Du a, Xinyuan Zhang a, Jian Song a, Yimei Yin a, He Huang a, Guanxin Shen b a b c
Department of Neurosurgery, Wuhan General Hospital of Guangzhou Command, Wuhan 430070, China Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China Department of Pharmacy, Hubei University of Chinese Medicine, Wuhan 430065, China
a r t i c l e
i n f o
Article history: Received 31 March 2011 Received in revised form 18 July 2011 Accepted 18 July 2011 Available online 3 August 2011 Keywords: Glioma Transferrin receptor Monoclonal antibody Nimustine Chemoimmunotherapy
a b s t r a c t Transferrin receptor (TfR) has been used as a target for antibody-based therapy of cancer. Anti-TfR antibody together with chemotherapeutic drugs has potential for cancer therapy. In this study, we investigated the in vitro anti-tumor effects of the anti-TfR monoclonal antibody (mAb), 7579, alone or in combination with Nimustine, a chemotherapeutic drug, on the gliomas cell lines U251 and U87MG. Our results indicated that 7579 alone dramatically down-regulated surface expression of TfR on tumor cells and induced S phase accumulation and apoptosis of tumor cells. Compared with 7579 or Nimustine used alone, the combination of 7579 with Nimustine demonstrated enhanced growth inhibitory effect on tumor cells. PI (Propidium iodide)/ Annexin V staining analyzed by FCM (flow cytometry) demonstrated that 7579 enhanced the cytotoxic effects of chemotherapeutic drug on tumor cells, indicating the therapeutic effect of 7579 was mediated mainly by promoting tumor cell necrosis. Using the median-effect/combination-index isobologram method, we further evaluated the nature of 7579/chemotherapeutic drug interactions. Synergistic interaction was observed for combination of 7579 with Nimustine. Our study provides additional evidence to develop combination therapies of anti-TfR mAbs-plus chemoimmunotherapy for gliomas. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Gliomas are the most frequent primary central nervous system (CNS) neoplasms, accounting for more than 50% of all brain tumors [1]. Despite advances in diagnostic and surgical techniques, the prognosis of these patients remains poor and the median survival of 12 months has not significantly changed during the last several years [2]. The latter is primarily due to the fact that gliomas are highly resistant to chemotherapy and radiotherapy [3]. Transferrin receptor (TfR, CD71) plays a crucial role in the cellular uptake of iron and in the regulation of cell growth [4]. In malignant tissues, TfR is expressed more abundantly than in normal tissues [5]. Therefore, TfR could be a relevant target for antibody-based therapies against tumors [6–8]. Due to the in vitro and in vivo success of anti-TfR antibody in tumor cell killing [9–11], a Phase I clinical trial of an IgA monoclonal anti-Tf receptor antibody 42/6 was conducted in 27 patients with advanced refractory cancer of different origins [12]. The
☆ There is no conflict of interest in our research. ⁎ Corresponding author. Tel.: + 86 2768878528; fax: + 86 2787212690. E-mail address: [email protected] (X. Wen). 1 These authors contributed equally and share the first author. 1567-5769/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2011.07.014
treatment was well tolerated, highlighting the safety of anti-TfR immunotherapy. In vitro combination therapy of an anti-TfR MAb, A24, and Zidovudine demonstrated improved cytotoxic effects on adult T-cell leukemia cells [13]. In vitro, A24 blocked proliferation of mantle cell lymphoma cells (MCL) by inducing mantle cell lymphoma cell apoptosis through caspase-3 and caspase-9 activation, either alone or synergistically with chemotherapeutic agents Adriblastin,Ara-C and Etoposide [14]. MAb A24 prevented tumor establishment and decreased the growth rate of pre-established MCL tumors in athymic nude mice. Therefore, A24-based therapies alone or in association with classic chemotherapies could provide a new alternative strategy against MCL, particularly in relapsing cases. In general, hematopoietic tumors are particularly suitable for treatment using TfR-targeting therapeutics since they express high levels of TfR and are more sensitive to the inhibitory effect of anti-TfR antibodies than other malignancies [15]. Nevertheless, elevated levels of TfR expression are widely observed on non-hematopoietic tumor cells and correlated with tumor grade and stage or poor prognosis [16–19]. Thus, TfR is also considered as an attractive target for nonhematopoietic tumor therapy. Trowbridge et al. reported that an antiTfR antibody could be efficient in inhibiting human melanoma xenograft growth in athymic mice [7]. In addition, combinations of two anti-TfR mAbs had anti-proliferative effects on breast cancer cells in vitro and in vivo [4]. To explore the combination use of anti-TfR
G. Xu et al. / International Immunopharmacology 11 (2011) 1844–1849
mAbs with chemotherapeutic drugs for cancer chemoimmunotherapy may be a promising strategy for glioma chemoimmunotherapy. Alkylating drugs are the oldest class of anti-cancer drugs which are still commonly in use, and they remain important in the treatment of several types of cancers. Nimustine (ACNU) is a chloroethylating agent which has been used either alone or in combination with other agents for the treatment of glioma [20]. 7579 is a murine monoclonal anti-human TfR IgG antibody made in our lab. It is a potent growth inhibitor of the erythroleukemia cell line K562 [21]. In this study, we investigated the in vitro effects of 7579 and Nimustine individually or in combination with each other on TfR overexpressing glioma cell lines and fresh glioma tissue, including the effects on apotosis, cell cycle arrest, TfR expression, and cell proliferation. The cell lines used were U251 which is a human glioblastoma cell line and U87MG which is a human malignant glioma cell line.
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1.1. Materials and methods 1.1.1. Cells, antibodies and chemotherapeutic drugs The glioma cell lines U251 and U87MG cells were purchased from the Shanghai Institute for Biological Science of the Chinese Academy of Science. Cells were maintained in DMEM supplemented with 10% fetal bovine serum and incubated at 37 °C in a humidified atmosphere containing 5% CO2. 7579, an anti-TfR MAb, was purified and characterized in our laboratory as described previously [21]. Antibody to β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-CD71 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Non-specific mouse IgG and horseradish peroxidase (HRP)-conjugated goat antimouse IgG antibodies were from Southern Biotech (Birmingham, USA). Chemotherapeutic Nimustine was purchased from Daiichi Sankyo (Japan).
Fig. 1. Transferrin receptor (TfR) expression in glioma tissues and glioma cells. (A) TfR staining in the tissues glioma tissues (b: grade II; c: grade III; d: grade IV) and normal brain tissues. (B) TfR staining in U251 and U87MG cell lines. (C) Western blot analysis of TfR expression in the tissue of glioma and normal brain. Data are presented as means ± SEM from three separate experiments. The average signal intensity was standardized to β-actin. The ratio of TfR expression to β-actin expression was shown. (D) Western blot analysis of TfR expression in U251 and U87MG cell lines. The average signal intensity was standardized to β-actin. The ratio of TfR expression to β-actin expression was shown. Data were shown as means ± SD of triplicate experiments.*p b 0.01.
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1.1.2. Sample and clinical information Normal tissues (n= 3) and brain tumors (n= 20) were obtained from glioma patients undergoing primary resection or biopsy of their tumor at the department of Neurosurgery, Wuhan General Hospital of Guangzhou Command (Wuhan, China). There are three grades: lowgrade astrocytomas (LGA; gradeII) (n= 8); anaplastic oligodendrogliomas (grade III) (n= 7); and glioblastoma multiforme (GBM; gradeIV) (n= 5). Collection of the human tissue specimens was approved by the appropriate ethics committee and was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. All persons gave informed consent prior to their inclusion in the study. 1.1.3. Immunohistochemical staining and evaluation Immunohistochemical analysis of TfR expression in tumor tissues and cell lines was performed with mouse anti-CD71 antibody as the primary antibody, and with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody as the secondary antibody. 1.1.4. Western blot analysis Western blot analysis of TfR expression in tumor tissues and cells was performed with mouse anti-CD71 antibody as primary antibody, and followed by horseradish peroxidase (HRP)-conjugated goat antimouse IgG antibody as the secondary antibody. The protein band density was quantified using the ImageJ software (Version 1.38x),
with β-actin as a loading control. The relative expression of TfR protein was expressed as the ratio of TfR expression to β-actin expression. 1.1.5. Cell apoptosis analysis U251 and U87MG cells (2 × 105) were placed in 6-well plates and treated with 7579 (100 μg/mL) alone, chemotherapeutic drugs (Nimustine) (40 μg/mL and 60 μg/mL) alone or the combinations of 7579 and Nimustine for 72 h. The same concentration of nonspecific mouse IgG (100 μg/mL) was used for isotype controls. Tumor cells were then washed with ice-cold PBS and incubated with a combination of 10 μg/mL FITC–Annexin V and 50 μg/mL PI (Roche Diagnostics, Mannheim, Germany) [22] at room temperature for 30 min. Analysis was performed by FCM (Becton–Dickinson LSR II). 1.1.6. Cell proliferation analysis U251 and U87MG cells (2 × 105) were grown in 6-well plates and treated with 7579 alone (100 μg/mL), Nimustine alone (U251: 40 μg/mL, U87MG: 60 μg/mL) or a combination of 7579 and Nimustine. The same concentration of nonspecific mouse IgG was used for isotype controls. Tumor cells were labeled with carboxyfluorescein diacetate (CFSE) at time zero and then grown for 72 hours with the agents noted above. The cellular concentration of CFSE is reduced by 50% with each cycle with a resultant change in flourscent color. After 72 h of culture tumor cells were washed with ice-cold PBS and analysed by flow
Fig. 2. Combined effects of 7579 and Nimustine on apoptosis of glioma cells. Apoptosis of U251 cells detected with FCM after treated with the 7579 and Nimustine as indicated, alone or in combination, for 72 h. (B) Apoptosis of U87MG cells detected with FCM after treated with the 7579 and Nimustine as indicated, alone or in combination, for 72 h. (C)The statistical analysis of apoptosis of U251 cells. (D)The statistical analysis of apoptosis of U87MG cells. Data represent the means and standard deviation of 4 independent experiments. *p b 0.01.
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cytometry. The data from 10,000 cells were collected and analyzed using ModiFit Software. 1.1.7. Cell cycle analysis U251 and U87MG cells (2× 105) were grown in 6-well plates and treated with 7579 alone (100 μg/mL), Nimustine alone (U251: 40 μg/mL, U87MG: 60 μg/mL) or the combinations of 7579 and Nimustine for 72 h. The same concentration of nonspecific mouse IgG was used for isotype controls. Tumor cells were harvested and fixed with 70% ethanol at 4 °C overnight, and then stained with 50 μg/mL PI for 30 min. Analysis was performed by FCM. The data from 10,000 cells were collected and analyzed using ModiFit Software. 1.1.8. Statistical analysis Statistical significance of the data was calculated by ANVOA or Student's t-test. A significance level of p b 0.01was chosen. Both analyses were carried out using SPSS 10.0 statistical software. 1.2. Results 1.2.1. TfR expression in glioma specimens and cell lines We examined the TfR expression in glioma specimens and normal cells by immunohistochemistry and western blot. We tested different types of glial tumors using tumor samples from LGA (grade II), anaplastic oligodendrogliomas (grade III), and GBMs (grade IV). The expression of TfR in these tumors was compared with that of normal cells from patients undergoing resection or biopsy. We found that TfR
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expression was up-regulated in glioma specimens and that the expression level was positively correlated with tumor grade (Fig. 1A). The glioma cell lines U251 and U87MG expressed high levels of TfR. 1.2.2. Apoptosis effects of 7579 and chemotherapeutic drug combination on glioma cells The apoptosis of cells after treatment with a combination of 7579 and Nimustine or each agent alone for 72 h was evaluated by FCM with PI-Annexin V staining. As shown in Fig. 2, both the combination of 7579 and Nimustine and each agent alone induced the death of tumor cells (U251: 53, 37, and 9%, (*p b 0.01), respectively and for U87MG: 47, 31 and 8%, (*p b 0.01), respectively). Thus, compared with Nimustine or 7579 when used alone, combination treatment with 7579 and Nimustine enhanced apoptosis of tumor cells and appeared to be slightly more than additive. 1.2.3. Proliferation inhibitory effects of 7579 and chemotherapeutic drug combinations with on glioma cells The proliferation of glioma cells after treatment with the combination of 7579 and Nimustine or each agent alone for 72 h was evaluated by FCM with CFSE staining. As shown in Fig. 3, the combination and each agent alone inhibited proliferation of tumor cells (U251: 5.4, 7.9, and 11.2, (*p b 0.01), respectively and for U87MG: 5.1, 7.6 and 8.4, (*p b 0.01), respectively). Thus, compared with Nimustine or 7579 alone, treatment with the combination of 7579 and Nimustine exhibited increased inhibition of proliferation of tumor cells.
Fig. 3. Combined effects of 7579 and Nimustine on proliferation of glioma cells. (A) Proliferation of U251 cells detected with FCM after treated with the 7579 and Nimustine as indicated, alone or in combination, for 72 h. (B) Proliferation of U87MG cells detected with FCM after treated with the 7579 and Nimustine as indicated, alone or in combination, for 72 h. (C) The statistical analysis of proliferation of U251 cells. (D) The statistical analysis of proliferation of U87MG cells. Data represent the means and standard deviation of 4 independent experiments. *p b 0.01.
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Fig. 4. Cell cycle phase distribution of glioma cells treated with the 7579 and Nimustine alone or in combination. (A) U251 cells were treated with 7579/Nimustine combinations or each agent alone for 72 h. DNA contents were measured by PI staining. (B) U87MG cells were treated with 7579/Nimustine combinations or each agent alone for 72 h. DNA contents were measured by PI staining. Histograms present one out of 4 independent experiments. (C) Cell cycle phase distribution of U251 cells treated with 7579/Nimustine combinations or each agent alone. (D) Cell cycle phase distribution of U87MG cells treated with 7579/Nimustine combinations or each agent alone. Data represent the means and standard deviation of 4 independent experiments. *p b 0.01.
1.2.4. Effects of 7579 and chemotherapeutic drug combination on the cell cycle of glioma cells Fig. 4 showed the cell cycle distribution of tumor cells after treatment with the combination of 7579 and Nimustine, or each agent alone for 72 h as evaluated by FCM with PI staining. Complete results are given in Fig. 4. Briefly, both agents alone decreased the number of cells in G1 and the combination provided and a further decrease that was essentially additive (12%, 19%, and 28%, respectively for U251 cells) with similar results on the U87MG cell line that appeared significantly more than additive for the combination (12%, 28%, and 54%, respectively). Cells in S phase were increased with cells in G2 showed inconsistent changes. 2. Discussion Most normal cells other than immature erythroid cells are low in transferrin receptor (TfR) expression. However, actively proliferating tumor cells exhibit high levels of TfR [23]. The growth inhibitory property of anti-TfR antibodies has been appreciated since the 1980s [24]. As for the mechanisms of anti-TfR antibody mediated growth inhibition, different antibodies have shown different modes of action in different models [25,26]. We have previously reported the construction of an intracellular antibody anti-TfR scFv-intrabody [5] and a human–mouse chimeric antibody D2C [21], which displayed a tumor-specifc distribution and had a strong antitumor effect. In this study, we showed that glioma specimens as well as cell lines exhibited abundant expression of TfR, and that expression of TfR correlated positively with tumor grade. These results suggested that TfR is a promising target for therapeutic strategy in gliomas. We showed that a murine monoclonal anti-human TfR IgG antibody 7579 induced S phase accumulation and apoptosis in 7579treated tumor cells with a resultant anti-proliferative effect. Further, combination treatment with 7579 and Nimustine was more effective
than either agent alone. These biological effects provide a basis for mAb/chemotherapeutic drug combination therapy, and also help explain the underlying mechanisms of combined efficacy. The blood brain barrier (BBB) is a bidirectional transport system [27] and enables the receptor-mediated transcytosis of either transferrin (Tf) or a TfR mAb from the blood to the brain [28]. The transferrin receptor (TfR) is expressed at both the BBB and the brain cell membrane (BCM) [29], and prior work in rats showed that the murine OX26 mAb against the rat TfR could deliver a PNA radiopharmaceutical to experimental brain tumors in vivo [30]. In the study by Lee et al., the 8D3 rat mAb to the mouse TfR [31] is used as a brain drug-targeting vector to enable imaging of gene expression in vivo in a transgenic mouse model of Huntington's disease (HD). Because our anti-TfR mAb (7579) is a murine monoclonal anti-human TfR IgG antibody it cannot permeate through the BBB of a mouse, so it cannot be tested in a mouse model. In conclusion, we analyzed the in vitro therapeutic effects of 7579 and Nimustine alone and in combination with each other on glioma cells. The results suggest that combination therapy was more effective than the use of either agent alone. Our study helps clarify the mechanism of action of TfR-directed therapies, suggesting the addition of this targeted approach to currently available cancer treatments might be a better therapeutic strategy. We declare that we have no conflict of interest. Acknowledgements This work was supported by Grant from the Hospital foundation of Wuhan General Hospital of Guangzhou Command (NO. 201010). References [1] Kleihues P, Louis DN, Scheithauer BW, Rorke LB, Reifenberger G, Burger PC, et al. The WHO classification of tumors of the nervous system. J Neuropathol Exp Neurol 2002;61:215–25.
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