Cancer Letters 302 (2011) 119–127 Contents lists available at ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet Adeno-associated virus-mediated anti-DR5 chimeric antibody expression suppresses human tumor growth in nude mice Fujia Lv, Yuhe Qiu, Yaxi Zhang, Shilian Liu, Juan Shi ⇑⇑, Yanxin Liu ⇑⇑, Dexian Zheng ⇑ National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, China a r t i c l e i n f o Article history: Received 17 November 2010 Received in revised form 10 January 2011 Accepted 12 January 2011 Keywords: Chimeric antibody Death receptor 5 Gene transfer Cancer therapy a b s t r a c t In the present study we demonstrate that adeno-associated virus (AAV)-mediated antiDR5 (death receptor 5) mouse–human chimeric antibody (shorten as Adximab) expression signiﬁcantly suppressed tumor cell growth by inducing apoptosis both in vitro and in vivo. The viral-expressed and cell-secreted Adximab efﬁciently bound DR5 with an afﬁnity of 0.7 nM and induced apoptosis of various tumor cells, but not normal cells. A single intramuscular injection of recombinant AAV particles resulted in a stable expression of Adximab in mouse serum for at least 70 days. AAV-mediated Adximab expression led to a signiﬁcant suppression of tumor growth in nude mice receiving xenografts of human liver and colon cancer. These data suggest that chimeric antibody gene transfer may provide an alternative strategy for the therapy of varieties of cancers. Ó 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Tumor necrosis factor-related apoptosis inducing ligand (TRAIL/Apo-2L), a member of the TNF family, can speciﬁcally induce cell death in various tumors but not in most normal cells and tissues . At least ﬁve TRAIL receptors have been identiﬁed in humans: TRAIL receptor 1 (death receptor 4, DR4), TRAIL receptor 2 (death receptor 5, DR5), TRAIL receptor 3 (decoy receptor 1, DcR1), TRAIL receptor 4 (decoy receptor 2, DcR2) and osteoprotegerin (OPG). Both DR4 and DR5 contain an intracellular death domain (DD), which triggers cell death signaling via activation of the caspase cascade and cleavage of downstream caspase substrates [2–4]. The other three receptors – DcR1, DcR2 and OPG – lack an intact death domain and are unable to induce apoptosis, but they compete with DR4 and DR5 for binding to TRAIL [5–7]. Upon TRAIL stimulation, death receptors (DRs) rapidly recruit a ⇑ Corresponding author. Tel.: +86 10 65296409; fax: +86 10 65105102. ⇑⇑ Co-corresponding author. Tel.: +86 10 65296409; fax: +86 10 65105102. E-mail address: [email protected] (D. Zheng). 0304-3835/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2011.01.001 death-inducing signaling complex (DISC), which consists of a Fas-associated death domain (FADD), procaspase-8 or -10, and/or receptor interaction protein (RIP) . Therefore, procaspase-8 or -10 is cleaved into caspase-8 or -10, which further activates the downstream procaspase-3, -6 and -7 . This is known as the DR-mediated extrinsic signaling pathway. Bcl-2 interacting domain (Bid) could also be cleaved by caspase-8 in certain types of cells, leading to release of cytochrome c (Cyt C) from mitochondria and formation of the apoptosome complex of procaspase-9, Apaf1 and Cyt C, followed by successive activation of procaspase-9, -3, -6 and -7 . This is known as the DR-mediated intrinsic signaling pathway. DR5 expression is frequently detected on tumor cell lines and various clinical cancer specimens, whereas there is limited expression of DR5 in normal cells and tissues . In mice, DR5 deﬁciency enhances lymph node metastasis without affecting primary tumor development , suggesting that DR5 plays an essential role in immune surveillance of malignancy under normal physiological conditions. Recently, it was reported that inﬂuenza and HIV-1 virus infection increased DR5 expression in NK cells, T lymphocytes, and monocytes or monocyte-derived 120 F. Lv et al. / Cancer Letters 302 (2011) 119–127 dendritic cells [13,14]. Also DR5 could induce monocytemediated tumor cell apoptosis . These data indicate that DR5 plays a critical role in innate immunity. However, the physiological role of DRs is not yet well understood. Since DRs are expressed in a variety of normal cells and tissues, toxicity to normal cells is a concern when DRs are targeted. Indeed, some reports demonstrated toxicity to normal hepatocytes, keratinocytes and neutrophils when DR’s were triggered by recombinant TRAIL in vitro [16– 18]. However, Lawrence et al. demonstrated that the reported cytotoxic effect of recombinant TRAIL on normal human liver cells was due to the tag linked with TRAIL, given that no cytotoxicity was seen with non-tagged TRAIL . A speciﬁc agonistic antibody against the death receptor is considered to be a safer and more effective cancer therapy than DR ligands. Several speciﬁc antibodies against DR4 or DR5 have been tested in clinical trials with promising outcomes [20–24]. At present, the immunogenicity of mouse mAbs can be successfully reduced by generating murine remodeling antibodies (mouse–human chimeric antibody or humanized antibody), which have been clinically validated for use in a wide variety of applications in immune therapy. The remodeling antibody has several advantages, including an antigen binding afﬁnity similar to the intact parental murine antibody, the ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) by the human Fc domain, which may enhance the therapeutic effect [25,26]. Despite the advantages of remodeling antibodies, antibody production, puriﬁcation and half life in vivo still create a bottleneck for both preclinical study and clinical use. To overcome these problems, antibody gene therapy has been demonstrated to be a viable therapeutic strategy for cancer and other chronic diseases. There are three major factors to consider in gene therapy: the transgene carrier, controllable transgene expression, and safety. Considering the matter of the transgene carrier, adeno-associated virus (AAV) vectors have been recognized as having distinct advantages, including long-term transgene expression, low immunogenicity and more safety in vivo . Guo et al. previously reported that a novel mouse antihuman DR5 monoclonal antibody, AD5–10, induced apoptosis in various tumor cells with no toxicity to normal hepatocytes or primary peripheral blood lymphocytes in vitro. Systemic administration of AD5–10 in nude mice with implanted human liver or lung cancer signiﬁcantly inhibited the tumor formation and growth without causing toxicity to the liver, spleen or kidney . These results suggest that AD5–10 is a promising agonistic antibody for cancer therapy. However, murine monoclonal antibodies can induce the human anti-mouse antibody (HAMA) response . To circumvent this problem, Shi et al. performed an antibody gene therapy study using AAVmediated expression of the single-chain Fv fragment (scFv) of AD5–10 in transformed cell lines as well as human tumor-bearing mouse models . The results showed that viral expression of the scFv antibody induced signiﬁcant apoptosis in various tumor cells and prevented tumor growth in the animal models. Since the life span of the small molecule scFv in vivo is limited, in this study we further established an AAV-mediated human-mouse chimeric antibody expression system by fusing the VH and VL cDNAs of AD5–10 with the heavy and light chain of human Fc, respectively. An anti-DR5 human–mouse chimeric antibody (Adximab) was successfully expressed both in cell lines and animals by transformation with the expression vectors or infection with the recombinant viral particles. The expression, afﬁnity and tumoricidal activity of the chimeric antibody were examined, and the results demonstrated that AAV-mediated Adximab expression signiﬁcantly suppressed tumor cell growth by inducing apoptosis both in vitro and in vivo. This study provides further insight into the antibody gene therapy for clinical application. 2. Materials and methods 2.1. Cell lines and culture Human embryonic kidney cell line HEK293, breast cancer cell line MDA-MB-231, epithelial carcinoma cell line Hela, colon cancer cell line HCT116, and lung cancer cell line A549 were purchased from American Type Culture Collection (Manassas, VA). Human glioma cancer cell line U251 and liver cancer cell line SMMC7721 were purchased from the Institute of Cell and Biochemistry, Chinese Academy of Sciences (Shanghai, China). Human colon cancer cell line COLO205 was purchased from the Cell Culture Center, Institute of Basic Medical Sciences (Beijing, China), and human embryonic eye Tenon’s ﬁbroblast cell line HFTF from the Cell Bank, Chinese Academy of Sciences (Shanghai, China). These cells were cultured in RPMI 1640, DMEM, or L-15 (Gibco, Grand Island, NY, USA) at 37 °C in an atmosphere of 5% CO2 incubator. The entire medium was supplemented with 10% heat-inactivated fetal calf serum (Hyclone, Logan, UT, USA), 100 units/ml penicillin, and 100 lg/ml streptomycin. 2.2. Constructs and generation of rAAV2 particles VH and VL cDNAs of the chimeric antibody Adximab were generated from the murine antibody AD5–10 . The VH and VL cDNAs were linked with the Fc of human IgG heavy and light chain to form the constructs Adximab heavy chain (Adximab-HC) and Adximab light chain (Adximab-LC), respectively. The Adximab-HC and Adximab-LC fragments were subcloned into an adeno-assoicated virus serotype 2 (AAV2) expression vector pAM-CAG (cytomegalovirus enhancer plus chicken b-actin promoter) . Enhanced green ﬂuorescent protein (EGFP) subcloned into the AAV2 vector was as the control. The generated expression vectors were depicted as pAM-CAG-Adximab-HC, pAM-CAG-Adximab-LC, and pAM-CAG-EGFP, respectively. Recombinant AAV viral particles (rAAV) were generated in HEK293 cells using a three-plasmid packing system, including one therapeutic plasmid and two helper plasmids H22 and pFd6 . The puriﬁcation of rAAV particles was performed by heparin afﬁnity column chromatography. The titers of rAAV particles were mea- F. Lv et al. / Cancer Letters 302 (2011) 119–127 sured by real-time PCR and presented as virus genomes/ml (vgs/ml). 2.3. Transfection and infection HEK293 cells (5 105) were cultured in 6-well plates and co-transfected with pAM-CAG-Adximab-HC and pAM-CAG-Adximab-LC plasmids using lipofectamine 2000 (Invitrogen, CA, USA). The cells and cell medium were collected separately at the indicated time for later on analysis. The cells transfected with pAM-CAG-EGFP plasmid were used as control. For rAAV infection, HEK293 cells (5 105) were cultured in 6-well plates in completed DMED medium for at least 8 h, then rAAV-Adximab-HC (5 104 vgs per cell) and rAAV-Adximab-LC (5 104 vgs per cell) particles were added and cultured in DMED without fetal bovine serum for an additional 6–8 h. The medium was switched to fresh complete DMED medium and cultured for 96 h. The medium was then collected for the analysis of Adximab concentration by ELISA. The cells infected with rAAV-EGFP (5 104 vgs per cell) were used as negative control. 2.4. Cell viability assay Cells (1 104) were cultured in 96-well plates for at least 8 h and treated with Adximab secreted from plasmids co-transfected HEK293 cells medium for 24 h. The cell viability was observed under a light microscope and evaluated by 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyltetrazolium (MTT) assay according to the manufacturer’s instructions (Sigma, St. Louis, MO, USA). 2.5. Western blot analysis One hundred lg of total protein sample from cell or tissue lysate, and 10 ll of the cell culture medium were subjected to a 12% SDS–PAGE. The separated proteins in the gel were transferred to a polyvinylidene diﬂuoride (PVDF) membrane (Amersham Biosciences, Little Chalfont, UK). The membrane was then incubated with speciﬁc primary 121 antibodies and HRP-conjugated secondary antibodies, successively. Primary antibodies were directed against caspase-8 (Oncogene, La Jolla, CA, USA), caspase-3 and caspase-9 (Cell Signaling, Beverly, MA, USA); poly ADP-ribose polymerase (PARP) (BD, San Jose, CA, USA); b-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), human IgG Cj and horseradish peroxidase (HRP) linked antihuman IgG Fc (Sigma, St. Louis, MO, USA). HRP linked antimouse, anti-rabbit or anti-goat IgG secondary antibodies were purchased from Zhongshan Biotech (Beijing, China).The proteins of interest were visualized by using the ECL chemiluminescence system according to the manufacturer’s instructions (Amersham Biosciences). 2.6. ELISA Ninety-six-well plates were coated with anti-human IgG (H + L) chain antibody (KPL, Gaithersburg, Maryland) (1:200 dilutions) and blocked with 5% nonfat dry milk. Then medium or mouse serum was incubated in the wells at indicated dilutions. The concentration of Adximab was detected by following incubation with HRP-conjugated anti-human IgG Fc antibody. The absorbance was measured at 492 nm on a microtiter reader (Thermo absystems, Finland). The concentration of Adximab in the cell medium or mouse serum was calculated according to the standard curve using normal human IgG. To measure the afﬁnity of Adximab binding to human DR5, 96-well plates were coated with recombinant soluble DR5 (3 lg/ml)  and blocked with 5% nonfat dry milk. Then the cell medium was added at indicated dilutions and incubated for 2 h, followed by incubation with HRPconjugated anti-human IgG Fc antibody. The absorbance was measured at 492 nm on a microtiter reader (Thermo absystems, Finland). The data were analyzed using GraphPad Prism Software. 2.7. Animal study 4- to 6-week-old male BALB/c nude mice were purchased from the Institute of Animal Sciences, Chinese Fig. 1. Expression of Adximab in HEK293 cells. (A) Schematic diagram of the recombinant expression plasmids of pAM-CAG-Adximab-HC and pAM-CAGAdximab-LC. HC, heavy chain; LC, light chain. VH, variable region of heavy chain; VL, variable region of light chain; CH1, CH2, CH3, constant region 1, 2, and 3 of heavy chain; CK, constant region of light chain (j chain). (B) Expression of Adximab in HEK293 cells analyzed by Western blot. The cells were cotransfected with the recombinant plasmids of pAM-CAG-Adximab-HC and pAM-CAG-Adximab-LC. Ten microlitres of the cell culture media were collected at indicated time points and subjected to a 12% SDS–PAGE followed by Western blot assay by using speciﬁc antibodies against human HC or LC, and horseradish peroxidase (HRP)-conjugated secondary antibody, respectively. The cells transfected with pAM-CAG-EGFP were used as control. (C) Expression of Adximab secreted in the cell culture medium. The cell culture media were collected at the indicated time points and subjected to a Sanwich ELISA to determine the concentration of Adximab by using the anti-human IgG (H + L) antibody as capturer and the HRP-labeled anti-human IgG Fc antibody as the second antibody. The data are expressed as the mean from at least three independent experiments. 122 F. Lv et al. / Cancer Letters 302 (2011) 119–127 Academy of Medical Science (Beijing, China) and housed under speciﬁc pathogen-free conditions. SMMC7721 tumor xenograft mouse model was established by subcutaneously injecting human liver cancer cells SMMC7721 (2 106 cells per mouse) into the right dorsal ﬂank. When the tumor size reached about 50 mm3, the animals were divided into two groups (n = 5) based on the tumor size. rAAV-Adximab (1 1011 particles) were injected into tumors in each mouse in the experimental group, while an equal amount of rAAV-EGFP particles were injected into tumors in each mouse in the control group (n = 5). Tumor size was measured every 4 days over 32 days and calculated by the following formula: volume = (ab2)/2 (a: the longest axis, b: the shortest axis). HCT116 tumor xenograft mouse model was established by s.c. inoculation of human colon cancer cells HCT116 (5 106 cells per mouse) into the right dorsal ﬂank. The mice were divided into four groups (n = 6–8) when the tumor size reached about 100 mm3. High dose of 1 1011 vgs and low dose of 2 1010 vgs of rAAV-Adximab and rAAV-EGFP particles were injected into the tumors in the experimental and control groups, respectively. Tumor size was monitored every 4 days during a period of 32 days. At the end of the experiment, the tumor was surgically excised and weighed. For the in vivo expression study, 1 1011 vgs of rAAVAdximab particles were injected into the muscle of BALB/ c nude mice, and an equal amount of rAAV-EGFP particles were injected as negative control. The blood samples were collected once a week during a period of 70 days. The expression of Adximab in mouse serum was analyzed by ELISA. At the end of the experiment, the animals were sacriﬁced and tissues were surgically excised for Adximab expression assay by Western blot or histochemical analysis. 2.8. In situ cell apoptosis analysis Parafﬁn-embedded sections of tumor tissues were preﬁxed in 4% paraformaldehyde and dewaxed. Cell apoptosis was detected by using TUNEL (terminal deoxynucleotidyl Fig. 2. Adximab efﬁciently binds DR5 and induces cell death by apoptosis in tumor cells. (A) The binding afﬁnity of Adximab with DR5 determined by ELISA. Wells of 96-well plate were coated with the recombinant soluble human DR5 protein, and incubated with culture medium of HEK293 cells transfected with the recombinant plasmids or infected with rAAV-Adximab particles (5 104 vgs per cell) for 4 days. Horseradish peroxidase (HRP)-conjugated anti-human IgG Fc antibody was used as the second antibody. The data are expressed as the mean from three independent experiments. (B) Cytotoxicity of Adximab in various tumor cell lines. HCT116, SMMC7721, Hela, A549, MDA-MB-231, COLO205, U251, and HFTF cells were cultured in the 4th day’s medium (100 ll per well) collected from HEK293 cells co-transfected with pAM-CAG-Adximab-HC and pAM-CAG-Adximab-LC. The cell viability was measured by MTT assay. The data are expressed as the mean from three independent experiments. (C) Cytotoxicity of Adximab in HCT116 and SMMC7721 cells treated for 24 h with the 4th day’s medium collected from the HEK293 cells co-transfected with pAM-CAG-Adximab-HC and pAM-CAG-Adximab-LC and observed under light microscope (magniﬁcation, 200). (D) Caspase activation in HCT116 and SMMC7721 cells analyzed by Western blot. The cells were treated for 24 h with the medium collected from HEK293 cells co-transfected with pAM-CAG-Adximab-HC and pAM-CAG-Adximab-LC and lysed with lysis buffer. The lysates were subjected to SDS–PAGE followed by Western blot with the speciﬁc antibodies against caspase-8, caspase-9, caspase-3 and the caspase substrate PARP. GAPDH was used as protein loading control. F. Lv et al. / Cancer Letters 302 (2011) 119–127 123 transferase-mediated dUTP nick end labeling) assay according to the manufacturer’s instructions (Roche, Mannheim, Germany). Apoptotic cells in the deﬁning regions of interest (ROI) were quantiﬁed by using automated cell acquisition and the software for immunohistochemistry (Histoquest software, TissueGnostics GmbH, Vienna, Austria). 2.9. Statistical analysis All data were expressed as mean value ± standard deviation (SD), and the statistical differences between groups were evaluated using Student’s t test. P < 0.05 was considered to be signiﬁcant. 3. Results 3.1. Expression of Adximab in HEK 293 cells Expression of the heavy chain (HC) and light chain (LC) of the chimeric antibody Adximab was ﬁrst performed in HEK293 cells co-transfected with the recombinant AAV vectors of pAM-CAG-Adximab-HC and pAMCAG-Adximab-LC (Fig. 1A). Western blots with antibodies against the HC and LC of human IgG, respectively, showed that the soluble form of Adximab was secreted into the medium of HEK293 cells in a time-dependent manner (Fig. 1B) and the concentration of Adximab, as determined by ELISA, reached 11.03 ± 0.19 lg/ml on the fourth day post-transfection (Fig. 1C), indicating that the chimeric antibody Adximab was expressed in HEK293 successfully. 3.2. Adximab efﬁciently binds DR5 and induces apoptosis in various tumor cell lines To test the ability of Adximab to bind to DR5, an ELISA was performed in the wells of a 96-well plate coated with recombinant soluble human DR5 protein, and binding was detected with HRP-conjugated anti-human IgG Fc antibody. As shown in Fig. 2A, the afﬁnity of Adximab from the medium of recombinant plasmid-transfected or rAAV particle-infected HEK293 cells was 0.9 nM or 0.7 nM, respectively. To test the cytotoxicity of Adximab to various tumor cell lines, the cells of HCT116 (colon cancer), SMMC7721 (liver cancer), Hela (cervical carcinoma), A549 (lung cancer), MDA-MB-231 (breast cancer), COLO205 (colon cancer), U251 (glioma) and HFTF (human embryonic eye Tenon’s ﬁbroblast cells) were cultured for 24 h in medium (100 ll per well) collected from a 4-day culture of HEK293 cells co-transfected with recombinant plasmids of pAM-CAGAdximab-HC and pAM-CAG-Adximab-LC, and the cell viability was measured by MTT assay. As shown in Fig. 2B, the viabilities of HCT116 and SMMC7721 were dramatically reduced to 34.96 ± 4.76% and 33.56 ± 17.33%, respectively, and the viabilities of A549, MDA-MB-231, COLO205, U251 and Hela cells were also reduced, to different levels. Importantly, the viability of the normal cell HFTF was unaffected by Adximab, indicating that Adximab is speciﬁcally cytotoxic to tumor cells, but not to normal cells. Morphological observation under a light microscope showed many dead cells in tumor cell cultures treated with chimeric antibody (Fig. 2C). In addition, caspase activation analysis by Western blot showed a decrease in full-length procaspase-3, -8, and -9, as well as an increase in cleavage of downstream caspase substrate PARP (Fig. 2D), suggesting that the chimeric antibody induces tumor cell death by caspase activation. Collectively, these data indicate that AAV-mediated Adximab expression efﬁciently binds DR5 and induces caspase-dependent apoptosis speciﬁcally in various tumor cell lines. 3.3. AAV-mediates long-term gene expression of Adximab To test the effects of stable Adximab expression in animals, 1 1011 vgs of recombinant AAV-Adximab viral particles were injected into the muscle of BALB/c nude mice. An equal amount of recombinant AAV-EGFP viral particles was used as control. Blood samples were collected once a week over 70 days to analyze the expression of Adximab by ELISA. As Fig. 3. Long-term expression of Adximab in BALB/c nude mice. (A) Concentration of Adximab in the serum of mouse i.m. injected with rAAVAdximab particles. The serum was collected at indicated time and analyzed by ELISA using anti-human IgG Fc antibodies. The serum of mouse i.m. injected with rAAV-EGFP were used as control. (B) Expression of Adximab in the mouse muscle detected by Western blot analysis. The mouse muscle tissue was collected on the 70th day post-infection with rAAV-Adximab or rAAV-EGFP particles and lysed with lysis buffer. The lysate was subjected to SDS–PAGE followed by Western blot assay using the speciﬁc antibodies against human HC and LC, respectively. Adximab expression was representative of three specimens examined and two of them were showed. The expression of GAPDH was used as protein loading control. shown in Fig. 3A, Adximab concentrations in the mouse serum reached 0.41 lg/ml on the 14th day post-infection, then increased to 1.5 lg/ml on the 40th day and remained unchanged until the end of the experiment. Western blot analysis at the end of the experiment conﬁrmed signiﬁcant expression of Adximab in the injected muscle (Fig. 3B). Tissues including heart, liver, spleen, lung, kidney and muscle were surgically excised from the animals. The hematoxylin–eosin staining demonstrated that there were no pathological changes in these tissues (photograph not shown). Together, these data substantiate that recombinant AAV particles could mediate long-term Adximab expression in vivo without systemic toxicity. 3.4. AAV-mediated Adximab expression suppresses tumor growth To investigate the efﬁcacy of chimeric antibody gene therapy for cancers, two human tumor xenograft models were established in BALB/c nude mice by subcutaneously inoculating SMMC7721 liver cancer and HCT116 colon cancer cells into the right dorsal ﬂanks. When the tumor size reached 50–100 mm3, the animals were divided into two groups based on tumor size, and rAAV-Adximab and rAAV-EGFP viral particles (1 1011 vgs) were injected into the tumors. The tumor size was measured every 4 days for a period of 32 days. The expression of Adximab in the mice was examined by Western blot. As shown in Fig. 4A, Adximab was highly expressed in the rAAV viral particle-injected SMMC7721 and HCT116 xenografts. In the SMMC7721 xenograft model, the mean tumor volume of the experimental group was 682 ± 204 mm3, whereas that of the control group was 1821 ± 220 mm3 (Fig. 4B), suggesting that AAVmediated Adximab gene therapy in mouse markedly suppresses SMMC7721 tumor growth (p = 0.00003, p < 0.01). In the HCT116 124 F. Lv et al. / Cancer Letters 302 (2011) 119–127 Fig. 4. Adximab expression suppressed human tumor growth in nude mice. (A) Western blot analysis of Adximab-HC and -LC expression in SMMC7721 and HCT116 xenograft mice on the 32nd day post-infection with rAAV-Adximab. The animals were sacriﬁced and the tumor tissues were surgically excised. The tumor tissues were smashed and lysed with lysis buffer. The lysate containing 100 lg of total protein was subjected to SDS–PAGE followed by Western blot assay. Adximab expressions were representatives of 3–6 specimens examined and one of SMMC7721 and 3 of HCT116 tumor tissues were showed. The rAAV-EGFP was used as negative control. The b-actin and GAPDH were used as protein loading controls. (B) rAAV-Adximab infection suppressed SMMC7721 tumor growth. The cells (2 106 cells per mouse) were inoculated into the right dorsal ﬂanks of the mice. When the tumor size reached about 50 mm3, the animals were divided into two groups (n = 5) based on the tumor size and rAAV-Adximab particles were injected into the tumor. rAAV-EGFP injection was used as control. Tumor volumes were measured every 4 days during a period of 32 days. (C) rAAV-Adximab infection suppressed HCT116 tumor growth. The cells (5 106 cells per mouse) were inoculated into the right dorsal ﬂanks of the mice. When the tumor size reached about 50 mm3, the animals were divided into four groups (n = 6–8) based on the tumor size. High dose (1 1011 vgs) and low dose (2 1010 vgs) of rAAV-Adximab particles were injected, respectively. Equal dose of rAAV-EGFP was used as control. Tumor volumes were measured every 4 days during the period of 32 days. (D) HCT116 tumor weight at the end of the experiment. ⁄⁄p < 0.01. xenograft model, two experimental groups with injections of 1 1011 vgs (high dose) and 2 1010 vgs (low dose) per mouse were performed. As shown in Fig. 4C, the mean tumor volume was reduced from 5198.81 ± 1691.44 mm3 to 3059.20 ± 562.15 mm3 (p = 0.01478, p < 0.05) in the high-dose group and from 4515.84 ± 820.54 mm3 to 2482.49 ± 393.06 mm3 (p = 0.00002, p < 0.01) in low-dose group. The tumor weight at the end of the experiment was reduced from 4.30 ± 0.97 g to 2.74 ± 0.49 g (p = 0.0056, p < 0.01) in the high-dose group and from 3.77 ± 0.92 g to 2.07 ± 0.32 g (p = 0.0002, p < 0.01) in the lowdose group (Fig. 4D). Taken together, these data demonstrate that AAVmediated Adximab expression in mice suppressed tumor growth signiﬁcantly. To determine whether the tumoricidal activity of AAV-mediated Adximab expression results in tumor cell death by apoptosis in vivo, TUNEL analysis was carried out in tumor tissue sections. As shown in Fig. 5A, a substantial number of apoptotic cells were detected in SMMC7721 liver tumor injected with rAAV-Adximab particles, whereas few were observed in the tumor injected with rAAV-EGFP control particles. A similar phenomenon was conﬁrmed in HCT116 colon tumor injected with high dose of rAAV-Adximab particles. After quantiﬁcation by automated cell acquisition and Histoquest software, TUNEL mean intensities in SMMC7721 liver tumor injected with rAAV-Adximab particles was 81.20 ± 3.70%, injected with rAAV-EGFP was 38.65 ± 3.04% (p = 0.0001, p < 0.01). In HCT116 tumor tissue, the TUNEL mean intensities injected with rAAVAdximab particles was 55.19 ± 6.84%, whereas injected with rAAV-EGFP was only 20.25 ± 5.50% (p = 0.0023, p < 0.01) (Fig. 5B, C). Thus, these data suggest that AAV-mediated Adximab antibody gene expression efﬁciently suppresses human tumor growth in nude mice by inducing cell death by apoptosis. 4. Discussion We have demonstrated in the present study that adenoassociated virus-mediated anti-DR5 mouse–human chimeric antibody (Adximab) gene expression signiﬁcantly suppressed human tumor growth both in vitro and in vivo. The afﬁnity of the viral expressed Adximab to DR5 was 0.7–0.9 nM, similar to 0.3 nM of the parental murine antibody AD5–10 , but signiﬁcantly higher than 4 nM of the viral expressed scFv of AD5–10 , suggesting that the AAV-expressed chimeric antibody adequately retained the properties of the parental murine monoclonal antibody. The cell-secreted Adximab to DR5 induced cell death in various tumor cells by apoptosis, but not in the normal cells of HFTF, and triggered the activation of caspase -3, -8, and -9 and the cleavage of PARP, the substrate of F. Lv et al. / Cancer Letters 302 (2011) 119–127 125 Fig. 5. Apoptotic cell death in rAAV-Adximab infected tumor tissue. (A) Parafﬁn-embedded sections of SMMC7721 and HCT116 tumor tissues were prepared from the mice at the end of the experiment. Apoptotic cells in the tumor tissues were visualized by TUNEL assay. Brown colored cells represent apoptotic cells. rAAV-EGFP was used as control (magniﬁcation, 400). (B) Apoptotic cells in the deﬁning regions of interest (ROI) were quantiﬁed by using automated cell acquisition and the software for immunohistochemistry (Histoquest). (C) TUNEL mean intensities in tumor tissue from the representative animal infected with rAAV-Adximab and rAAV-EGFP, respectively. ⁄⁄p < 0.01. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) caspases. The tumoricidal activity of Adximab was conﬁrmed in vivo. Single injections of recombinant AAV viral particles in mouse muscle resulted in long-term (at least 70 days) and therapeutic expressions of the chimeric antibody in mouse serum and led to a remarkable suppression of tumor xenograft growth. However, it was notably that there was no signiﬁcant differences in the ability of high (1011 vgs) and low (2 1010 vgs) doses of viral Adximab to inhibit HCT116 tumor growth. This result suggests that that the viral dose might differ by more magnitudes, which is remained to be evaluated. The viral expressed chimeric antibody might be more effective as a therapeutic than the scFv reported by Shi et al.  because the chimeric antibody may have a longer half life in vivo. The improved tumoricidal activity of Adximab versus the parental murine antibody indicates that the chimeric antibody may elicit a more effective immunological response than the scFv fragment. Li et al. (unpublished data) have conﬁrmed that the chimeric antibody induced antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC), consistent with reports by Sánchez-Mejorada et al.  and Nechansky et al. . Importantly, AAV-mediated antibody gene expression for the therapy of cancer and other chronic diseases could resolve several serious bottlenecks, such as time, costs and techniques involved in producing and manufacturing reshaped monoclonal antibodies, because this strategy permits long-term antibody gene expression in vivo, leading to signiﬁcant inhibition of tumor growth in human liver cancer SMMC7721 and colon cancer HCT116 in xenograft mouse models. Comparing with recombinant antibody, 126 F. Lv et al. / Cancer Letters 302 (2011) 119–127 which half life in vivo is usually from several days to no more than 4 weeks , AAV-medaited antibody expression, which could be last at least for 70 days, may much superior to administration of the puriﬁed recombinant antibodies. However, several important problems need to be resolved before antibody gene transfer becomes an alternative supplement to the current therapies. First, expression of the chimeric antibody following gene transfer has to be controllable because certain normal cells may express the autologous DR5, which could be targeted by the chimeric antibody and lead to autoimmune conditions. Second, the immunogenicity of chimeric antibodies during long-term expression has to be clariﬁed, because treatment with inﬂiximab, a chimeric monoclonal IgG1 antibody against tumor necrosis factor, has been reported to result in the formation of antibodies against inﬂiximab . 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