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

The clinical use of the single-chain fixed-variable (scFv) fragments of recombinant monoclonal antibodies as credible alternatives for classic therapeutic antibodies has two limitations: rapid blood clearance and inefficient local expression of functional molecules. In attempt to address these issues, we have developed a novel gene therapy protocol in which the anti-death receptor 5 (DR5) scFv fragments were either in vitro expressed in several tumor cell lines, or in vivo expressed in mice, using recombinant adeno-associated virus (rAAV)–mediated gene transfer. Viral transduction using the rAAV-S3C construct, which encodes a scFv molecule (S3C scFv) specific to DR5, led to stable expression in tumor cell lines and showed apoptosis-inducing activity in vitro , which could be inhibited by recombinant DR5 but not by DR4. A single i.m. injection of rAAV-S3C virus in nude mice resulted in stable expression of DR5-binding S3C scFv proteins in mouse sera for at least 240 days. Moreover, the expression of S3C scFv was associated with significant suppression of tumor growth and the increase of tumor cell apoptosis in previously established s.c. human lung LTEP-sml and liver Hep3B tumor xenografts. (Cancer Res 2006; 66(24): 11946-53)


Introduction
Death receptor 5 [DR5, or tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptor 2], a member of the TNF receptor superfamily, is emerging as a favorable target for the design of anticancer agents in recent years (1).When triggered by its ligand, DR5 undergoes oligomerization, mediated by its cytoplasmic death domains, and propagates apoptotic signaling cascades through nucleating the formation of death-inducing signaling complexes (2)(3)(4)(5).DR5 can be specifically activated by TNF-related apoptosis-inducing ligand (TRAIL), which also activates another functional death receptor, DR4 (or TRAIL receptor 1).Upon triggering, DR5 selectively induces cell death in a wide variety of tumor cells both in vitro and in vivo under different experimental settings (4)(5)(6)(7).However, at least certain versions of recombinant soluble TRAIL (sTRAIL) are also shown to cause apoptosis in normal cells (especially in hepatocytes), hampering its clinical use for cancer therapy (8,9).As an alternative, agonistic monoclonal antibodies (mAb) specifically targeting DR5 are raised for the purpose of cancer treatment (10,11).We previously reported a novel mouse anti-human DR5 mAb, AD5-10, which stimulated apoptosis in several tumor cell lines in vitro in the absence of cross-linking and showed strong tumoricidal activity in vivo with negligible cytotoxicity toward normal cells (12).
A critical drawback of using mouse antibodies on patients, however, is associated with the human immunoreactions to murine antibodies, which makes long-term use of this therapy infeasible for patients with chronic progressive conditions such as cancers (13).The immunogenicity of rodent mAbs sometimes can be successfully reduced, but in a laborious and time-consuming way, by antibody chimerization and humanization using proteinengineering technologies.In addition, due to the large size of intact antibodies, they are difficult to penetrate into solid tumors, further limiting their applications in immunotherapy (14).
Recently, the development of small recombinant antibody fragments as high-affinity therapeutic reagents with reduced immunogenicity has come under the spotlight (15)(16)(17).A popular format of engineered recombinant antibody fragment is the singlechain fixed-variable (scFv) molecule, in which the V H and V L regions of the parental antibody are joined by a polypeptide linker.The scFv fragment retains the target specificity and antigenbinding affinity of the intact antibody, whereas it can be genetically designed and produced in large quantity by ectopically expressing both V H and V L regions from a single cDNA in cells (18,19).Moreover, due to its smaller size, the scFv molecule shows improved pharmacokinetics in tumor penetration and is better tolerated by the host immune system (20,21).The length of the linker peptide seems to play an important role in affecting the function of scFv by determining whether the scFv molecules exist as monovalent monomers, or autoassemble into divalent dimmers (diabodies) and trivalent trimers (tribodies; refs.[22][23][24][25][26][27][28][29]. Despite the many advantages of using scFv molecules for 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 high local concentrations of operative molecules at the target site (13).To overcome these problems, techniques to in vivo express therapeutic antibodies or their fragments have been developed.Among them, recombinant adeno-associated virus (rAAV)-based vectors are recently shown to be a credible gene carrier for in vivo gene expression in a variety of gene therapy applications.The advantages of using rAAV-based vectors include long-term transgene expression and low immunogenicity (30).
In this study, we developed a gene therapy protocol in which our previously reported mouse anti-human DR5 mAb, AD5-10, was engineered into the scFv format and introduced into s.c.human lung LTEP-sml and liver Hep3B tumor xenografts for in vivo expression via rAAV vector-mediated gene transfer.Xenografts i.m. injected with rAAV-S3C, a rAAV carrying a cDNA that encodes the anti-DR5 scFv (S3C scFv) molecule, exhibited sustained serum level of DR5-binding S3C scFv (100-200 Ag/mL) for at least 240 days.The virally expressed scFv antibody fragments stimulated apoptosis in tumor cells and inhibited tumor growth in mice.

Materials and Methods
Animals.All surgical procedures and care administered to the animals were approved by the University of Hong Kong Ethics Committee and done according to institutional guidelines.BALB/c male nude mice, 4 to 5 weeks old, were housed at a constant temperature and supplied with laboratory chow and water ad libitum on a 12 hours:12 hours light/dark cycle.
Cells.Human lung cancer cell line A549 (lung adenocarcinoma), human liver cancer cell lines Hep3B and HepG2, and human colon cancer cell line HCT116 were originated from the American Type Culture Collection (Rockville, MD).Human liver cancer cell lines SMMC-7721 and BEL7402 were supplied by the Institute of Cell and Biochemistry, Chinese Academy of Sciences (Shanghai, China).Human small lung cancer cell line LTEP-sml was kindly provided by Professor Xiaoguang Chen (Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China).These cells were cultured at 37jC in RPMI 1640, F12, or DMEM (Life Technologies, Grand Island, NY).The primary human hepatocytes (PHH), kindly provided by the Department of Surgery, University of Hong Kong, were maintained in Waymouth's MB-752/1 medium (Life Technologies) supplemented with 2 mmol/L glutamine, 150 nmol/L insulin, 10% FCS, 100 units/mL penicillin, 100 Ag/mL streptomycin, and 100 nmol/L dexamethasone, and were plated at a density of 5 Â 10 4 cells/cm 2 .
Construction of the recombinant rAAV-S0C, rAAV-S1C, rAAV-S3C, rAAV-SIRESC vectors, and rAAV vectors encoding sTRAIL and enhanced green fluorescent protein.The CAG promoter, the inserted genes, and polyadenylic acid sequences were inserted between the inverted terminal repeats of an AAV plasmid using appropriate restriction enzymes.A woodchuck hepatitis posttranscriptional regulatory element (WPRE), which was shown to boost transcription, was inserted into the constructs immediately after the expression cassette (31).The cDNA of AD5-10 was used as the template for the scFv fragments.V H and V L sequences of AD5-10 were cloned and linked with a linker sequence to form a scFv ''gene'' in the V H -linker-V L orientation.S0C was the scFv construct encoding the V H and V L domains that are directly linked.In S1C and S3C, the V H domain was separated from the V L domain by a linker sequence encoding a polypeptide containing 5 amino acids (GGGGS) and 15 amino acids (GGGGSGGGGSGGGGS), respectively.In the SIRESC construct, sequence encoding V H domain was separated from V L domain by an internal ribosome entry site to ensure the expression of these two domains from the same RNA template.The scFv constructs with different linkers were subcloned into the AAV plasmid and subsequently packaged and purified.Recombinant AAV vectors encoding sTRAIL (rAAV-sTRAIL) or enhanced green fluorescent protein (rAAV-eGFP) were constructed as previously described (32).
Production of rAAV particles.AAV serotype 2 (AAV2) was used for in vitro studies.For in vivo experiments, however, a pseudotyping strategy was used to produce rAAV particles packaged with AAV1 capsid proteins (AAV2/1).This is because AAV1 is more efficient in muscle transduction and evokes less host immune reaction.Infective rAAV particles were generated by a three-plasmid, helper virus-free packaging method (32).The titer of rAAVs was quantified by real-time PCR analysis.
Apoptosis and viability determination.Cell apoptosis was determined by propidium iodide staining and flow cytometry assay.In brief, cells were harvested by trypsinization at the indicated hours after treatment and stained with 50 Ag/mL propidium iodide.The tubes were placed at 4jC in the dark for 0.5 hour before flow cytometry analysis.The propidium iodide fluorescence of individual nuclei was measured in the red fluorescence and the data were registered in a logarithmic scale.At least 10 4 cells of each sample were analyzed.Apoptotic nuclei appeared as a hypodiploid DNA peak before the G 1 phase of cell cycle, which was easily distinguished from hyperdiploid DNA peak of cells at the G 1 phase or later at the G 2 and S phases.Cell viability was quantified by 3-(4,5-dimethyl-thiazole-2-yl)-2,5-biphenyl tetrazolium (MTT) assay (Sigma, St. Louis, MO; ref. 32).
Membrane protein extraction.The cell membrane fraction was 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 EGTA, 10 mmol/L leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride].The homogenate was spun at 86,000 Â g at 4jC for 45 minutes.The pellet (membrane fraction) was resuspended in homogenization buffer containing 1% Triton X-100 and left on ice for 30 minutes, followed by centrifugation at 14,000 Â g at 4jC for 30 minutes.The supernatant from this spin was the solubilized membrane fraction.
Detection of DR5 by flow cytometry assay.Phycoerythrin-labeled antihuman DR5 antibody (R&D Systems, Minneapolis, MN) was used to detect the DR5 expression level on the cell surface.The assay was carried out according to the manufacturer's instruction.Briefly, 10 5 cells were added into phycoerythrin-conjugated antibodies after first blocking with human IgG and incubated for 30 minutes at 4jC.After washing twice with PBS, cell surface expression of TRAIL receptors was determined by flow cytometry using 488-nm-wavelength laser excitation.The phycoerythrin-labeled mouse IgG 2B (R&D Systems) was used as control.
Analysis of transgene expression.The expressions and activities of transgenes were verified by using standard techniques, including Western blot analysis, immunohistochemical staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, as previously described (32).The antibodies against XIAP, cIAP1, caspase-3, and caspase-8 were from Santa Cruz Biotechnology (Santa Cruz, CA).
S3C scFv concentration determination.The S3C scFv concentration in the medium and mouse sera was determined by ELISA.The purified recombinant S3C scFv protein expressed in Escherichia coli was used as standard.Briefly, 96-well plates were coated for 2 hours at 37jC with 200 AL/well of recombinant S3C protein (1 ng/mL-10 Ag/mL, 1:5 serial dilution), or medium and mouse sera prediluted (1:10-1:500) with 0.05 mol/L Na 2 CO 3 /NaHCO 3 (pH 9.6).After washing with PBS, the plates were blocked with the blocking buffer (0.05% Tween 20 and 5% nonfat milk in PBS) at 37jC for 0.5 hour, and followed by incubation at 37jC for 2 hours with an anti-AD5-10 antibodies raised in rabbit using a synthetic high variable region peptide of AD5-10 heavy chain.After washing, the horseradish peroxidase (HRP)-conjugate goat anti-rabbit IgG and 1,2-benzenediamine (0.4 mg/mL, Sigma) was sequentially added and the color was allowed to develop at room temperature for 0.5 hour.The absorbance was measured at 490 nm on a SPECTRAMAX 340 microplate reader (Molecular Devices, Menlo Park, CA).
Analysis of DR5 binding by S3C scFv.S3C scFv expressed in HEK293 cells transduced with the rAAV-S3C vector or in the sera of the mice injected with rAAV2/1-S3C were detected.To evaluate binding activity of S3C to human DR5, the plates were coated with 200 ng/well of recombinant DR5 protein (12), blocked with 5% nonfat dry milk, and incubated with various concentrations of S3C scFv or AD5-10.After sequential incubation with rabbit anti-AD5-10 antibodies and HRP-conjugated goat antirabbit antibodies, the bindings were visualized by adding the peroxidase substrate.The absorbance was measured at 490 nm on a microplate reader.The data were analyzed by using GraphPad Prism software (GraphPad Software, San Diego, CA).
The S3C scFv binding to DR5 was also determined by coimmunoprecipitation.Medium from HEK293 cell culture transduced with rAAV-S3C for 4 days were incubated at 37jC for 3 hours with 100 ng/mL homemade recombinant human DR5.Anti-AD5-10 antibody and protein A Sepharose were then added and the coprecipitated proteins were resolved with SDS-PAGE and probed for DR5.
For immunoprecipitation of native DR5 in cells, HCT116 cells were incubated with the medium of rAAV-S3C-transduced HEK293 cells.Cells were lysed in the buffer [30 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1% NP40, 10% glycerol] supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany).After 15 minutes of incubation on ice, the lysates were centrifuged at 20,000 Â g and 4jC for 15 minutes.Approximately 700 to 800 Ag of total protein were incubated with the anti-AD5-10 antibody followed by immunoprecipitation with magnetic protein A. The coprecipitated proteins were subjected to SDS-PAGE and DR5 immunoblotting analysis.
S.c.tumor xenografts and assessment of tumor growth.The Hep3B cells (1 Â 10 6 in 100 AL PBS) or LTEP-sml cells (5 Â 10 6 in 100 AL PBS) were injected in the s.c.tissue of the right dorsal flank of 6-week-old male nude mice.Tumor growth was monitored twice a week by measuring tumor size with calipers.Tumor volume (V ) was calculated using the formula V = 1/2 Â length Â (width) 2 .When the tumors had reached f50 mm 3 in size, animals were divided into two groups (n = 6-10) and the indicated rAAV vectors were administered by i.m. injection.Group 1 received rAAV2/ 1-S3C at a dose of 1 Â 10 11 genome equivalents.Group 2 received empty rAAV2/1 at a dose of 1 Â 10 11 genome equivalents.Experiments were terminated when tumors in the control group showed signs of necrosis.
Statistical analysis.Results were expressed as mean values F SD. Student's t test was used to evaluate statistical significance.P < 0.05 was considered significant.

Results
Construction and expression of scFv fragments.The scFv constructs were generated by using the cDNA encoding the V H and V L chains of AD5-10 as the template.As previously mentioned, the length of the linker may affect the function of the scFv molecules.We thus constructed four AD5-10 scFv cDNAs, in which the V H and V L encoding sequences were either directly linked (S0C scFv cDNA) or separated by oligonucleotides encoding for different lengths of linker peptides (S1C, S3C, and SIRESC scFv cDNAs).The S1C and S3C scFv cDNAs expressed S1C and S3C scFv molecules with a 5-amino-acid (GGGGS) and a 15-amino-acid linker (GGGGSGGGGSGGGGS), respectively.In SIRESC scFv cDNAs, an internal ribosome entry site element was inserted between V H and V L cDNAs to promote the expression of V H and V L domain independently (Fig. 1A).The scFv cDNA fragments were then cloned into a rAAV vector.The recombinant virus was prepared by a three-plasmid, helper virus-free packaging method (32).The resulting viruses were added into HEK293 cell cultures for infection.Western blot using an antibody specific for V H domain of AD5-10 showed that the rAAV scFv constructs were expressed at a similar level in infected cells (Fig. 1B).
Cytotoxicity of the S3C scFv fragments among various tumor cell lines.The cytotoxic activities of these scFv fragments were tested in several tumor-derived cell lines.Each cell line was infected with equal numbers of viral particles [multiplicity of infection (MOI) = 5 Â 10 4 ] for 24, 48, or 96 hours.At each time point, the viability of infected cells was determined by MTT assay.As shown in Fig. 2A, transduced expression of the rAAV-S3C vector resulted in cell growth inhibition in most cell lines tested, including the human lung cancer cell lines A549 and LTEP-sml, liver cancer cell lines HepG2 and Hep3B, and colon cancer cell line HCT116.As a comparison, the extent of viability reductions induced by the rAAV-S0C, rAAV-S1C, or rAAV-SIRESC transduction resembled that of the rAAV-eGFP infection in these cells (P < 0.05 in A549 cell line and P < 0.01 in other four tumor cell lines, 96 hours).Similar results were observed in human liver cancer cell lines SMMC 7721 and BEL 7402 (data not shown).Notably, the rAAV-S3C construct showed stronger inhibitory activity than rAAV-sTRAIL in Hep3B and LTEP-sml cells (P < 0.01, 96 hours), suggesting that the activity of rAAV-S3C may be dependent on cell type.The cytotoxicity of rAAV-S3C seemed to be specific for tumor cell lines because the viability of the PHHs were unaffected by virus infection (Fig. 2A).
Because the parental antibody of S3C scFv, AD5-10, had been reported to induce apoptosis in tumor cell lines in a DR5dependent manner, we then analyzed the DR5 expression in the membrane fraction of cell lysates prepared from above cell lines by immunoblotting.As shown in Fig. 2B, all tumor cell lines expressed the DR5 protein, although at different levels.Interestingly, no membrane DR5 was detectable in PHH cells (Fig. 2B), and forced overexpression of DR5 in PHH cells (Fig. 2C) failed to sensitize PHH cells to S3C scFv-induced cell death (Fig. 2D) even at high MOI.
To determine whether the S3C scFv-mediated cell killing involves the function of DR5, we carried out a competitive experiment in vitro where increasing concentrations of recombinant DR5 or DR4 proteins were added into rAAV-S3C-or rAAV-eGFP-infected LTEP-sml cell culture.Relative cell viability was determined by the MTT assay.As shown in Fig. 2E, recombinant DR5 protein, but not DR4, protected LTEP-sml cells from the cytotoxicity of rAAV-S3C.A similar result was observed in rAAV-S3C-infected Hep3B cell culture (data not shown), suggesting the involvement of DR5 in S3C scFv-induced cytotoxicity.
Collectively, these data showed that the cytotoxicity of engineered AD5-10 scFv fragments toward tumor cell lines might be affected by length of the linker peptide.The rAAV-S3C construct, which encoded an anti-DR5 scFv fragment with a 15-amino-acid linker, was able to induce cell death via DR5 in a time-dependent manner in several cancer cell lines, but not in normal human hepatocytes.B, Western blot analysis of scFv expression in rAAV-scFv-infected HEK293 cells.Cells were infected with the indicated rAAV constructs at a MOI of 5 Â 10 4 and were collected 72 hours later.Cell lysates were subjected to SDS-PAGE and probed for scFv fragments using anti-AD5-10 antibodies.h-actin was also probed with specific antibodies for its use as the loading control.
S3C scFv induced tumor cell apoptosis via a classic apoptosis pathway.To determine if the cytotoxicity induced by rAAV-S3C-mediated gene expression was due to apoptosis, propidium iodide staining and flow cytometry analysis were done in rAAV-S3C-infected Hep3B and LTEP-sml cells.The hypodiploid DNA peaks, which indicate cell apoptosis, were seen in rAAV-sTRAIL-and rAAV-S3C-infected cells (Fig. 3A); the apoptosis rate of LTEP-sml cells 72 hours after transduction of rAAV-S3C was 30.37 F 8.14%, which was significantly higher than rAAV-sTRAILinfected LTEP-sml cells (P = 0.0034 versus rAAV-eGFP, P = 0.0089 versus rAAV-sTRAIL), and the apoptosis rate of rAAV-S3C-infected Hep3B cells was 27.91 F 4.95% (P = 0.0017 versus rAAV-eGFP, P = 0.027 versus rAAV-sTRAIL).Stimulation of DR5 usually induces the classic apoptosis pathway characterized by caspase-3 and caspase-8 activation, as well as simultaneous down-regulation of the inhibitor of apoptosis protein XIAP and cIAP1.Indeed, these were observed in tumor cell lines expressing S3C scFv fragments (Fig. 3B).These data showed that the cytotoxicity of S3C scFv in tumor cell lines was due to the activation of the classic apoptosis pathway mediated by death receptors.
Muscular expression of S3C scFv in mice.To express the S3C scFv in mice, a pseudotyped version of rAAV2/1 was created by using rep from AAV2 and cap from AAV1.The rAAV2/1 vector was selected for its low immunogenicity and efficient transduction in muscle cells (33,34).The rAAV2/1 virus was used for in vivo delivery of S3C scFv via i.m. injection into BALB/c nude mice.As shown in Fig. 4A, the expression of S3C scFv was easily detectable in isolated mouse muscle sections of 50 days after injection of 1 Â 10 11 genome equivalents of the rAAV2/1-S3C virus.Immunoblotting analysis further confirmed S3C scFv expression in vivo in injected muscle tissues (Fig. 4B).
The virally expressed S3C scFv fragments were released in a soluble form into the animal sera, which can be readily determined by ELISA, with a peak level at 133.37 F 26.33 Ag/mL 60 days after injection (Fig. 4C).With only a slight decline after 60 days, the serum level of scFv seemed to be stable and was maintained at approximately 100 to 120 Ag/mL throughout the 8 months' study.
Taken together, these results showed that i.m. injection of rAAV2/1-S3C virus induced stable expression of S3C scFv fragments in systemic circulation.
Secreted S3C scFvs efficiently bound to DR5.Next, we tested if the virally expressed S3C scFv fragments in the medium of infected cells and the sera of transduced mice maintained its affinity toward its antigen.Immobilized DR5 protein in a 96-well plate was incubated with the medium of rAAV-S3C-infected HEK293 cells or the sera of rAAV-S3C-transduced mice.The binding affinity between the two proteins was measured by ELISA.The results showed that serum S3C scFv efficiently bound to its antigen (K d = 4 Â 10 À9 mol/L).When compared with that of the intact antibody (K d = 10 À10 mol/L), the DR5 binding affinity of the antibody fragment was slightly lower, but a little higher than that of the S3C scFvs secreted to the medium of virus-infected HEK293 cells (K d = 3.3 Â 10 À9 mol/L; Fig. 5A).To further confirm the binding of S3C scFv to DR5, recombinant DR5 protein was added into medium prepared from rAAV-S3C virus-infected HEK293 to induce the formation of S3C scFv-DR5 complex, which was, in turn, coimmunoprecipitated using the anti-AD5-10 antibody.As shown in Fig. 5B, the DR5 was coimmunoprecipitated with the antibody fragments only in rAAV-S3C-transduced cells.To show the recognition of native membrane DR5 by secreted S3C scFv antibody fragments, the cell culture medium from rAAV-S3Ctransduced HEK293 cells were added into HCT116 or the PHH cultures.S3C scFv were then immunoprecipitated with the anti-AD5-10 antibody.Coimmunoprecipitants were resolved on SDS-PAGE and probed for DR5.As shown in Fig. 5C, the native DR5 was able to form a stable complex with S3C scFv in HCT116 cells.The S3C-DR5 complex could not be found in PHH cells, which express undetectable level of membrane DR5.
These results showed that the secreted S3C scFv antibody fragment showed similar binding affinity as its parental antibody toward recombinant and native DR5 protein.
S3C scFv suppressed the growth of s.c.tumor xenografts by inducing tumor cell apoptosis.To analyze the therapeutic potential of the S3C scFv fragments, we established a tumor model in BALB/c nude mice by s.c.inoculation of human lung cancer LTEP-sml cells (5 Â 10 6 per injection) in the right dorsal flank of the mice.When the tumors had reached a size of f50 mm 3 , animals were divided into two groups (n = 6) and received an i.m. injection of 1 Â 10 11 particles of rAAV2/1-S3C or empty rAAV2/1 as control.The lung tumor volume was measured twice a week over a period of f50 days.The result showed that the mean tumor volume, which was 455 F 86 mm 3 in the control group, was reduced to 249 F 62 mm 3 in mice injected with rAAV2/1-S3C, demonstrating a substantial 45% decrease (P < 0.01) in tumor growth (Fig. 6A).
In another xenograft model, we s.c.implanted 1 Â 10 6 of human liver cancer Hep3B cells into the mice to establish a liver tumor xenograft model.Again, the animals were divided into two groups (n = 8-10) and the effect of rAAV2/1-S3C on tumor growth suppression was similarly evaluated as in the human lung cancer xenograft model.The result showed that injection of rAAV2/1-S3C reduced the mean tumor volume from 3,061 F 306 mm 3 , as observed in empty vector-injected mice, to 1,788 F 297 mm 3 (P < 0.01), accounting for a 42% decrease in tumor growth (Fig. 6B).
To determine whether treatment with rAAV2/1-S3C led to apoptosis in tumor cells, TUNEL assays were carried out in lung tumor sections acquired from rAAV2/1-S3C-or empty vectorinjected mouse xenografts.Results showed that the expression of the S3C scFv antibody fragment was associated with an accumulation of apoptotic cells within the tumors, which was largely absent in control mice (Fig. 6C).
We also examined the histopathologic changes in lung, liver, spleen, and kidney of the animals used in this study to investigate the cytotoxicity of S3C scFvs toward normal tissues.No obvious lesions were found in any of the organs (data not shown).Moreover, the rAAV2/1-S3C-injected mice did not show any sign of systemic toxicity as estimated by body weight, gross appearance, and behavior.

Discussion
In the present study, we developed a novel therapeutic strategy by expressing an anti-DR5 scFv antibody fragment, S3C scFv, in vivo through rAAV-based, vector-mediated gene transfer.We showed that this scFv fragment was stably expressed in mice by The BALB/c nude mice were given a single i.m. injection of 1 Â 10 11 particles of rAAV2/1-S3C or empty rAAV2/1 vector.Muscle sections were prepared 50 days after injection and were used for immunohistochemical staining using a rabbit anti-AD5-10 antibody followed by HRP-conjugated goat anti-rabbit antibody, and visualized by 3,3 ¶-diaminobenzidine staining.Magnification, Â100.B, whole-cell lysates were prepared from muscles of virus-infected nude mice.The expression of S3C scFv protein were determined by immunoblotting using anti-AD5-10 antibodies.Each lane corresponds to an individual mouse.C, level of secreted S3C scFv in mouse serum.Sera from rAAV-S3C scFv-infected ( w ) or empty vector-infected (n) mice were collected at the indicated times.The level of S3C scFv in sera were determined by ELISA using anti-AD5-10 antibodies.Points, mean of three mice; bars, SD.The scFv antibody fragments became recently known as promising alternatives for intact antibodies for immunotherapy.The linker region that connects V H and V L chains may affect the activity of scFvs.A linker of 15 residues or more usually renders the scFv monomeric with similar antigen-binding affinity as the parental antibody (22,23).Reducing linker length leads to autoassembly of scFv monomers to form functional divalent dimmers (diabodies; refs.[24][25][26].When the linker is completely removed, scFv molecules exist as trimers (tribodies), which can be either trivalent or nonfunctional (noncognate V H /V L pair; refs.[27][28][29].In our study, the S3C scFv antibody fragment, which has a linker of 15 residues and is presumed to be monovalent, showed the highest apoptosis-inducing activity in several tumor cell lines, which can be blocked by soluble DR5 but not by DR4.This result is consistent with our previous observation that the parental AD5-10 mAb did not require secondary cross-linking for DR5 triggering (12).We speculate that the underlying mechanism may be involved in antibody-induced specific conformational changes that are sufficient to stimulate receptor aggregation.However, further studies are required to confirm this hypothesis.
DR5 was widely expressed in a variety of tumor cells, including melanoma cell lines and nearly all non-small cell lung cancer tumors (35,36).Several studies had shown that TRAIL sensitivity was positively related to the level of death receptors (37)(38)(39).However, that effect might be limited to certain cell types.For example, cell lines such as LTEP-sml, Hep3B, and certain  melanoma cell lines were relatively resistant to sTRAIL despite moderate receptor expression and/or absence of decoy receptors (40).In this study, although all of the five tumor cell lines expressed different levels of surface DR5, their sensitivities toward S3C scFvinduced cell death were not proportional to the level of DR5 expression.Therefore, cells may incorporate additional regulatory signaling pathways to control DR5 receptor functions in a cell type-specific manner.Indeed, multiple layers of regulation have been suggested during TRAIL receptor activation (41)(42)(43).
The PHH cells did not express detectable membrane DR5 and were resistant to the killing by S3C scFvs.However, forced overexpression of DR5 in PHH cells failed to sensitize PHH cells to S3C scFv-induced cell death.As previously discussed, although overexpression of death receptors often enhances apoptosis in cells (44), it is not always the case (40).For example, despite similar surface expression of TRAIL receptor, primary and transformed keratinocytes exhibited marked difference in TRAIL sensitivity (45).Thus, sensitivity to receptor stimulation could be determined by the concerted regulations of multiple mechanisms including the level and availability of DR5, the balance between decoy receptors and death receptors, and distinct intracellular signaling pathways (40)(41)(42)(43).Moreover, the mechanism underlying AD5-10-mediated cell death pathway is not yet fully characterized and requires further analysis in details.Nevertheless, the resistance of PHH to S3C scFv-mediated cytotoxicity provided initial data for the safety of using rAAV-S3C in gene therapy.
We also showed that the i.m. injection of rAAV2/1-S3C virus in tumor xenografts led to stable serum secretion of S3C scFv fragments that induced apoptosis of implanted tumors.Systemic gene delivery carries the risk of gene delivery to other tissues such as liver.Therefore, it is highly desirable to achieve tissue-specific gene transfer and expression.In our study, we used the rAAV2/1 vector system, which has been shown to be more selective and safe for muscle transduction via i.m. administration (46).Transduction of rAAV2/1-S3C was confirmed by PCR amplification of a portion of the WPRE fragment from genomic DNA isolated from muscle biopsies at the site of injection.As shown in Supplementary Fig. SA, the DNA fragment encoding for WPRE was not detectable in liver, heart, spleen, lung, and kidney.Meanwhile, the expression of S3C scFv mRNA in the muscle was only found in the rAAV-S3C virustransduced mice, as determined by reverse transcription-PCR analysis (Supplementary Fig. SB).The absence of virus sequence in tissues other than muscle was consistent with the established properties of AAV2/1-based vectors.Therefore, the high musclespecific expression of S3C scFv achieved in vivo via rAAV2/1mediated gene transfer would make this vector potentially suitable for human use.
The apoptotic effect of S3C scFv antibody fragments on tumor cells in vivo and in vitro can only be described as moderate, although the concentrations of soluble S3C scFv protein secreted into the culture medium and serum were relatively high.One possible explanation might be the relatively low affinity of the fragment compared with the parental antibody.Protein engineering on AD5-10 is now underway in our laboratory to improve the binding affinity of both intact antibody and its fragments, which may enhance the apoptosis-inducing activity of the antibodies.In addition, increased therapeutic efficacy can be expected by combined use of rAAV-S3C virus with conventional therapeutics, such as chemotherapeutic agents and irradiation.
This study took advantage of our specific anti-DR5 (AD5-10) mAb and combined the scFv fragment technique with rAAV vector-mediated gene transfer to achieve tumor suppression in mice, providing a novel strategy with significant clinical prospect for gene therapy.

Figure 1 .
Figure1.Construction and expressions of rAAV-scFv constructs.A, schematic illustration of anti-DR5 scFv-expressing cassettes.SP, signal peptide.G4S, linker with the amino acid sequence GGGGS, where G is glycine and S is serine.B, Western blot analysis of scFv expression in rAAV-scFv-infected HEK293 cells.Cells were infected with the indicated rAAV constructs at a MOI of 5 Â 10 4 and were collected 72 hours later.Cell lysates were subjected to SDS-PAGE and probed for scFv fragments using anti-AD5-10 antibodies.h-actin was also probed with specific antibodies for its use as the loading control.

Figure 2 .
Figure 2. S3C scFv-stimulated tumor cell death involved DR5 function.A, cytotoxicities of different rAAV-scFv constructs on various tumor cell lines.The A549, LTEP-sml, Hep3B, HepG2, and HCT116 cells were infected with rAAV-scFv virus at a MOI of 5 Â 10 4 for the indicated time.Cell viability was determined by MTT assay.Columns, mean of three independent experiments with three replicates; bars, SD. *, P < 0.05; **, P < 0.01.B, immunoblot analysis of membrane DR5 expression in various tumor cells.Membrane fractions containing equal amounts of total protein were prepared from the whole-cell lysates and were subjected to SDS-PAGE, followed by immunoblotting with an anti-DR5 antibody.C, surface DR5 expression in the PHH cells.PHHs were transfected with pRK-DR5 expression vector and stained with an anti-DR5 antibody followed by flow cytometry analysis.Black filled area, pRK-DR5-transfected PHHs.Open area, wild-type PHHs (DR5 negative).Gray filled area, negative control (murine IgG 2B ).D, unsusceptibility of DR5-positive PHHs to S3C scFv-mediated apoptosis.pRK-DR5-transfected PHH cells were treated with the indicated virus at MOIs of 1 Â 10 4 to 1 Â 10 6 .Cell viability was determined by MTT assay.Columns, mean of three independent experiments with three replicates; bars, SD.E, DR5, but not DR4, blocked the cytotoxicity of S3C scFv by competition.Indicated concentrations of recombinant DR5 or DR4 proteins were added into rAAV-S3C-or rAAV-eGFP-infected LTEP-sml cells.Relative cell viability was determined by MTT assay.Representative of three independent experiments.

Figure 4 .
Figure 4. S3C scFv expression in mice.A, muscular expression of S3C scFv.The BALB/c nude mice were given a single i.m. injection of 1 Â 10 11 particles of rAAV2/1-S3C or empty rAAV2/1 vector.Muscle sections were prepared 50 days after injection and were used for immunohistochemical staining using a rabbit anti-AD5-10 antibody followed by HRP-conjugated goat anti-rabbit antibody, and visualized by 3,3 ¶-diaminobenzidine staining.Magnification, Â100.B, whole-cell lysates were prepared from muscles of virus-infected nude mice.The expression of S3C scFv protein were determined by immunoblotting using anti-AD5-10 antibodies.Each lane corresponds to an individual mouse.C, level of secreted S3C scFv in mouse serum.Sera from rAAV-S3C scFv-infected ( w ) or empty vector-infected (n) mice were collected at the indicated times.The level of S3C scFv in sera were determined by ELISA using anti-AD5-10 antibodies.Points, mean of three mice; bars, SD.

Figure 3 .
Figure 3. rAAV-S3C-mediated gene expression induced apoptosis in tumor cell lines.A, the Hep3B and LTEP-sml cells were infected by the indicated virus at a MOI of 5 Â 10 4 and analyzed 72 hours after infection by propidium iodide staining and flow cytometry.B, caspase activation and XIAP and cIAP1 inhibition in the virus-infected cells.LTEP-sml and Hep3B cells were infected with rAAV-S3C at a MOI of 5 Â 10 4 and harvested at the indicated time points.Approximately 50 Ag of total protein in the cell lysates were resolved by SDS-PAGE.Caspase-3 and caspase-8 activation and XIAP and cIAP-1 inhibition were detected by immunoblotting using corresponding antibodies.h-actin was used as the loading control.
i.m. injection and had tumor suppression activity in two s.c.human tumor xenograft models.

Figure 6 .
Figure 6.I.m.injection of rAAV2/1-S3C suppressed the growth of s.c.tumors in mice.A, LTEP-sml were injected in the s.c.tissue of the right dorsal flank of nude mice.When the tumors had reached a size of f50 mm 3 , animals were divided into two groups and i.m. injected with 1 Â 10 11 particles of rAAV2/1-S3C ( w ) or empty rAAV2/1 (n).Tumor volumes were determined as described in Materials and Methods.Points, mean of the tumor volume in six mice; bars, SD.B, tumor growth in the Hep3B tumor-bearing mice injected with either rAAV2/1-S3C ( w ) or the empty rAAV2/1 vector (n).Points, mean of the tumor volume in 8 to 10 mice; bars, SD.C, increased tumor cell apoptosis in S3C scFv-treated mice.LTEP-sml lung tumor sections were prepared from S3C scFv-treated mice and control mice.The apoptosis of tumor cells in these sections were determined by TUNEL staining (green ).Magnification, Â400.

Figure 5 .
Figure 5. S3C scFv formed complex with DR5.A, binding affinity of S3C scFv to DR5.Recombinant human DR5 protein of indicated concentrations was immobilized on a 96-well plate.The plate was then incubated with the AD5-10 antibody, culture medium from rAAV-S3C-infected HEK293 cells, or serum from mice infected with the virus for 50 days.Binding affinities of AD5-10 in vitro and in vivo secreted S3C scFv to DR5 were measured by ELISA using anti-AD5-10 antibodies.The absorbance was measured at 490 nm on a microplate reader.Representative of three independent experiments.B, the cell culture medium of HEK293 cells infected with rAAV-S3C or rAAV-eGFP virus for 4 days were collected and incubated with (+) or without (À) recombinant human DR5.Immunoprecipitation (IP ) was done by adding anti-AD5-10 antibodies and protein-A Sepharose beads.The coimmunoprecipitated proteins were subjected to SDS-PAGE and immunoblotting, and probed for DR5.C, recognition of native membrane DR5 by secreted S3C scFv antibody fragments.The cell culture medium of rAAV-S3C-or rAAV-eGFP-transduced HEK293 cells were added into HCT116 and PHH cultures.Endogenous S3C-DR5 complex were then immunoprecipitated with anti-AD5-10 antibodies from cell lysates.Coimmunoprecipitants were resolved on SDS-PAGE and probed for DR5.