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Therapeutic Expression of an Anti-Death Receptor 5 Single-Chain Fixed-Variable Region Prevents Tumor Growth in Mice

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Therapeutic Expression of an Anti-Death Receptor 5 Single-Chain Fixed-Variable Region Prevents Tumor Growth in Mice Research Article Therapeutic Expression of an Anti-Death Receptor 5 Single-Chain Fixed-Variable Region Prevents Tumor Growth in Mice Juan Shi,1 Yanxin Liu,1 Yong Zheng,2 Yabin Guo,1 Jinchun Zhang,1 Pik-to Cheung,3 Ruian Xu,1,4,5 and Dexian Zheng1 1National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; 2Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; 3Department of Pediatrics and Adolescent Medicine and 4Gene Therapy Laboratory, University of Hong Kong, Hong Kong, China; and 5Institute of Molecular Medicine, Huaqiao University, Quanzhou, China Abstract recombinant soluble TRAIL (sTRAIL) are also shown to cause The clinical use of the single-chain fixed-variable (scFv) apoptosis in normal cells (especially in hepatocytes), hampering its fragments of recombinant monoclonal antibodies as credible clinical use for cancer therapy (8, 9). As an alternative, agonistic alternatives for classic therapeutic antibodies has two monoclonal antibodies (mAb) specifically targeting DR5 are raised limitations: rapid blood clearance and inefficient local for the purpose of cancer treatment (10, 11). We previously expression of functional molecules. In attempt to address reported a novel mouse anti-human DR5 mAb, AD5-10, which these issues, we have developed a novel gene therapy protocol stimulated apoptosis in several tumor cell lines in vitro in the absence of cross-linking and showed strong tumoricidal activity in which the anti-death receptor 5(DR5)scFv fragments were either in vitro expressed in several tumor cell lines, or in vivo in vivo with negligible cytotoxicity toward normal cells (12). expressed in mice, using recombinant adeno-associated virus A critical drawback of using mouse antibodies on patients, (rAAV)–mediated gene transfer. Viral transduction using the however, is associated with the human immunoreactions to murine rAAV-S3C construct, which encodes a scFv molecule (S3C scFv) antibodies, which makes long-term use of this therapy infeasible specific to DR5, led to stable expression in tumor cell lines and for patients with chronic progressive conditions such as cancers showed apoptosis-inducing activity in vitro, which could be (13). The immunogenicity of rodent mAbs sometimes can be inhibited by recombinant DR5but not by DR4. A single i.m. successfully reduced, but in a laborious and time-consuming way, injection of rAAV-S3C virus in nude mice resulted in stable by antibody chimerization and humanization using protein- expression of DR5-binding S3C scFv proteins in mouse sera for engineering technologies. In addition, due to the large size of at least 240 days. Moreover, the expression of S3C scFv was intact antibodies, they are difficult to penetrate into solid tumors, associated with significant suppression of tumor growth and further limiting their applications in immunotherapy (14). the increase of tumor cell apoptosis in previously established Recently, the development of small recombinant antibody s.c. human lung LTEP-sml and liver Hep3B tumor xenografts. fragments as high-affinity therapeutic reagents with reduced (Cancer Res 2006; 66(24): 11946-53) immunogenicity has come under the spotlight (15–17). A popular format of engineered recombinant antibody fragment is the single- Introduction chain fixed-variable (scFv) molecule, in which the VH and VL regions of the parental antibody are joined by a polypeptide linker. Death receptor 5 [DR5, or tumor necrosis factor (TNF)–related The scFv fragment retains the target specificity and antigen- apoptosis-inducing ligand (TRAIL) receptor 2], a member of the binding affinity of the intact antibody, whereas it can be genetically TNF receptor superfamily, is emerging as a favorable target for designed and produced in large quantity by ectopically expressing the design of anticancer agents in recent years (1). When triggered both VH and VL regions from a single cDNA in cells (18, 19). by its ligand, DR5 undergoes oligomerization, mediated by its Moreover, due to its smaller size, the scFv molecule shows cytoplasmic death domains, and propagates apoptotic signaling improved pharmacokinetics in tumor penetration and is better cascades through nucleating the formation of death-inducing tolerated by the host immune system (20, 21). The length of the signaling complexes (2–5). DR5 can be specifically activated by linker peptide seems to play an important role in affecting the TNF-related apoptosis-inducing ligand (TRAIL), which also acti- function of scFv by determining whether the scFv molecules exist vates another functional death receptor, DR4 (or TRAIL receptor 1). as monovalent monomers, or autoassemble into divalent dimmers Upon triggering, DR5 selectively induces cell death in a wide variety (diabodies) and trivalent trimers (tribodies; refs. 22–29). of tumor cells both in vitro and in vivo under different Despite the many advantages of using scFv molecules for experimental settings (4–7). However, at least certain versions of immunotherapy, especially for chronic diseases, the treatment efficacy, however, is often compromised by the rapid blood clearance of infused scFv proteins and the difficulty in maintaining Note: Supplementary data for this article are available at Cancer Research Online high local concentrations of operative molecules at the target site (http://cancerres.aacrjournals.org/). J. Shi and Y. Liu contributed equally to this work. (13). To overcome these problems, techniques to in vivo express Requests for reprints: Dexian Zheng, National Laboratory of Medical Molecular therapeutic antibodies or their fragments have been developed. Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Among them, recombinant adeno-associated virus (rAAV)–based Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, China. Phone: 86- 10-6529-6409; Fax: 86-10-6510-5102; E-mail: [email protected] or Ruian Xu, vectors are recently shown to be a credible gene carrier for in vivo Institute of Molecular Medicine, Huagiao University, Quanzhuo, China. E-mail: gene expression in a variety of gene therapy applications. The [email protected] I2006 American Association for Cancer Research. advantages of using rAAV-based vectors include long-term trans- doi:10.1158/0008-5472.CAN-06-1227 gene expression and low immunogenicity (30). Cancer Res 2006; 66: (24). December 15, 2006 11946 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Prevention of Cancer Growth by Anti-DR5 scFv Expression In this study, we developed a gene therapy protocol in which our placed at 4jC in the dark for 0.5 hour before flow cytometry analysis. The previously reported mouse anti-human DR5 mAb, AD5-10, was propidium iodide fluorescence of individual nuclei was measured in the red fluorescence and the data were registered in a logarithmic scale. engineered into the scFv format and introduced into s.c. human 4 lung LTEP-sml and liver Hep3B tumor xenografts for in vivo At least 10 cells of each sample were analyzed. Apoptotic nuclei appeared as a hypodiploid DNA peak before the G phase of cell cycle, which expression via rAAV vector–mediated gene transfer. Xenografts i.m. 1 was easily distinguished from hyperdiploid DNA peak of cells at the G injected with rAAV-S3C, a rAAV carrying a cDNA that encodes the 1 phase or later at the G2 and S phases. Cell viability was quantified by anti-DR5 scFv (S3C scFv) molecule, exhibited sustained serum level 3-(4,5-dimethyl-thiazole-2-yl)-2,5-biphenyl tetrazolium (MTT) assay (Sigma, A of DR5-binding S3C scFv (100–200 g/mL) for at least 240 days. The St. Louis, MO; ref. 32). virally expressed scFv antibody fragments stimulated apoptosis in Membrane protein extraction. The cell membrane fraction was tumor cells and inhibited tumor growth in mice. obtained by homogenization and differential centrifugation. Briefly, cells were homogenized and sonicated on ice in homogenization buffer [20 mmol/L Tris-HCl (pH 7.4), 10% glycerol, 4 mmol/L EDTA, 2 mmol/L Materials and Methods EGTA, 10 mmol/L leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride]. The Animals. All surgical procedures and care administered to the animals homogenate was spun at 86,000 Â g at 4jC for 45 minutes. The pellet were approved by the University of Hong Kong Ethics Committee and done (membrane fraction) was resuspended in homogenization buffer containing according to institutional guidelines. BALB/c male nude mice, 4 to 5 weeks 1% Triton X-100 and left on ice for 30 minutes, followed by centrifugation at old, were housed at a constant temperature and supplied with laboratory 14,000 Â g at 4jC for 30 minutes. The supernatant from this spin was the chow and water ad libitum on a 12 hours:12 hours light/dark cycle. solubilized membrane fraction. Cells. Human lung cancer cell line A549 (lung adenocarcinoma), human Detection of DR5by flow cytometry assay. Phycoerythrin-labeled anti- liver cancer cell lines Hep3B and HepG2, and human colon cancer cell line human DR5 antibody (R&D Systems, Minneapolis, MN) was used to detect HCT116 were originated from the American Type Culture Collection the DR5 expression level on the cell surface. The assay was carried out (Rockville, MD). Human liver cancer cell lines SMMC-7721 and BEL7402 according to the manufacturer’s instruction. Briefly, 105 cells were added were supplied by the Institute of Cell and Biochemistry, Chinese Academy of into phycoerythrin-conjugated antibodies after first blocking with human Sciences (Shanghai, China). Human small lung cancer cell line LTEP-sml IgG and incubated for 30 minutes at 4jC. After washing twice with PBS, cell was kindly provided by Professor Xiaoguang Chen (Institute of Materia surface expression of TRAIL receptors was determined by flow cytometry Medica, Chinese Academy of Medical Sciences, Beijing, China). These cells using 488-nm-wavelength laser excitation. The phycoerythrin-labeled were cultured at 37jC in RPMI 1640, F12, or DMEM (Life Technologies, mouse IgG2B (R&D Systems) was used as control.
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