Enhancement of Antitumor Activity of Gammaretrovirus Carrying IL-12

ORIGINAL ARTICLE Enhancement of antitumor activity of gammaretrovirus carrying IL-12 gene through genetic modification of envelope targeting HER2 receptor: a promising strategy for bladder cancer therapy Y-S Tsai1,2,3, A-L Shiau1,4, Y-F Chen4, H-T Tsai3, T-S Tzai3 and C-L Wu1,5 1Institute of Clinical Medicine, National Cheng Kung University Medical College, Tainan, Taiwan; 2Department of Urology, National Cheng Kung University Hospital, Dou-Liou Branch, Yunlin, Taiwan; 3Department of Urology, National Cheng Kung University Medical College, Tainan, Taiwan; 4Department of Microbiology and Immunology, National Cheng Kung University Medical College, Tainan, Taiwan and 5Department of Biochemistry and Molecular Biology, National Cheng Kung University Medical College, Tainan, Taiwan

The objective of this study was to develop an HER2-targeted, envelope-modified Moloney murine leukemia virus (MoMLV)-based gammaretroviral vector carrying interleukin (IL)-12 gene for bladder cancer therapy. It displayed a chimeric envelope protein containing a single-chain variable fragment (scFv) antibody to the HER2 receptor and carried the mouse IL-12 gene. The fragment of anti-erbB2scFv was constructed into the proline-rich region of the viral envelope of the packaging vector lacking a transmembrane subunit of the carboxyl terminal region of surface subunit. As compared with envelope-unmodified gammaretroviruses, envelope- modified ones had extended viral tropism to human HER2-expressing bladder cancer cell lines, induced apoptosis, and affected cell cycle progression despite lower viral titers. Moreover, animal studies showed that envelope-modified gammaretroviruses carrying IL-12 gene exerted higher antitumor activity in terms of retarding tumor growth and prolonging the survival of tumor-bearing mice than unmodified ones, which were associated with enhanced tumor cell apoptosis as well as increased intratumoral levels of IL-12, interferon-g, IL-1b, and tumor necrosis factor-a proteins. Therefore, the antitumor activity of gammaretroviruses carrying the IL-12 gene was enhanced through genetic modification of the envelope targeting HER2 receptor, which may be a promising strategy for bladder cancer therapy. Cancer Gene Therapy (2010) 17, 37–48; doi:10.1038/cgt.2009.41; published online 19 June 2009 Keywords: HER2; interleukin-12; retrovirus; gene therapy; bladder cancer

4,5 Introduction course and a poorer response to chemotherapy in bladder cancers.6 Therefore, HER2-targeted strategies The human epidermal growth factor receptor (EGFR) have become very good potential therapies for HER2- 7,8 family is involved in signal transduction pathways expressing bladder cancers. resulting in cell proliferation, cell survival, angiogenesis, The minibody single-chain variable fragment (scFv), and metastasis in several human malignancies.1 HER2 in which the antigen-binding domains of an antibody (one member of the EGFR family, also called erbB2) (VH and VL) are joined by a flexible polypeptide linker, expression in urothelial cancers is variable, ranging from has considerable therapeutic potential against antigens 8.5 to 81%, but it has a higher frequency in invasive involved in disease processes, and through the process of bladder cancers.2,3 Some have reported a correlation antigen-antibody binding, it has the ability to specifically between HER2 overexpression, a more aggressive clinical target,9 with or without blocking signal transduction.10 De Lorenzo et al.11 reported that anti-erbB2scFv not only induced internalization but also displayed an intrinsic, Correspondence: Professor T-S Tzai, Department of Urology, strong, and specific anti-proliferative activity toward National Cheng Kung University Medical College, 1, Dashiue HER2-expressing cells. With such characteristics, the Road, Tainan 70101, Taiwan. E-mail: [email protected] or minibody has a promising potential for targeted cancer Professor C-L Wu, Department of Biochemistry and Molecular therapy. In contrast, Neve et al.12 showed that some anti- Biology, National Cheng Kung University Medical College, 1, Dashiue Road, Tainan 70101, Taiwan. erbB2scFvs exhibited only ErbB2-mediated endocytosis, E-mail: [email protected] without any correlation with the cell cycle, cell prolifera- Received 3 July 2008; revised 14 December 2008; accepted 24 tion, or intracellular kinase signal induction by inter- January 2009; published online 19 June 2009 nalizing antibodies. Enhanced killing of HER2-positive tumors by gammaretrovirus Y-S Tsai et al 38 Several investigators have used a modified viral carboxyl terminal domain of the surface glycoprotein envelope to enhance the specificity of infection by a (SU) of the MoMLV envelope resulted in a frame-shift chimera envelope to certain receptors, such as EGFR,13 mutation at the initial portion of the transmembrane cMET,14 and CEA.15 They constructed a new plasmid (TM) subunit. The coding region of anti-erbB2scFv was carrying the envelope sequence with insertion of scFv excised from the eukaryotic expression vector pMFverB9 cDNA into the gp70 of Moloney murine leukemia virus and inserted into the PRR of the envelope sequence of (MoMLV), such as the N-terminal receptor-binding pCL-Eco-DTM plasmid through the pMECA plasmid,7 domain (RBD) or the proline-rich region (PRR). Such a to generate pCL-Eco-anti-erbB2scFv (designated as pCL- strategy can exert an ability to specifically target EcoscFv). Furthermore, in frame fusion of the construct recombinant gammaretroviruses to certain receptors and was verified by sequencing (Figure 1). The coding regions then extend the gammaretroviral tropism. Despite this, of p35 and p40 subunits of IL-12 were obtained by PCR the therapeutic performance still relies on an efficient using adenoviral vector carrying mIL-12 (AdmIL-12, a transfer and expression of the gene of interest, which kind gift from FL Graham25) as the template. The might influence cell survival or proliferation.15 sequence of internal ribosome entry site (IRES) of Interleukin (IL)-12 has a unique 70-kDa-heterodimeric encephalomyocarditis virus (B600 bp) was also generated structure composed of two covalently linked p35 and p40 by PCR using pTCY-IRES-TK plasmid7 as the template, subunits, both of which are required for biological in which the IRES sequence was originally derived from activity.16 IL-12 exhibits many biological effects, includ- pIRES-hrGFP-1a (Stratagene, La Jolla, CA). The PCR ing a stimulatory effect on natural killer (NK) cells, the primers were 50-GGAATTCTTAGGCGGAGCTCAGA induction of cytotoxic T-cell activation, and the induction TAGC-30 (forward), 50-TTCGCCTCGAGTCTATCGCT of interferon (IFN)-g production,17,18 and possesses ACCTC-30 (reverse) for IL-12 p35, 50-CGAGCTCATGT antitumor, antiangiogenic, and antimetastatic activ- GTCCTCAGAAGCTAAC-30 (forward) and 50-GCGTC ities.19–21 IL-12 also has synergistic efficacy as an GACTTAGGATCGGACCCTGCAG-30 (reverse) for adjuvant for vaccination against cancers22,23 and can enhance HER2-specific protective and therapeutic antitumor immunity using genetically modified dendritic cells with HER2 antigen presentation.24 Therefore, this study was designed to investigate a new therapeutic strategy for HER2-expressing bladder cancer with envelope-modified gammaretroviruses displaying a scFv-targeted HER2 receptor and carrying the mouse IL-12 (mIL-12) gene. The synergistic effect of both minibody-tumor antigen binding and IL-12 expression was able to achieve an excellent therapeutic response in an immunocompetent syngeneic murine bladder tumor model.

Materials and methods Cell lines, culture conditions, and mice Murine fibroblasts (NIH3T3) and bladder cancer cell line (MBT2), as well as human embryonic kidney cell line (293 Figure 1 Schematic illustrations of the plasmid constructs. The cells) and bladder cancer cell lines (TSGH8301, J82, T24, coding region of the anti-erbB2scFv was inserted into retroviral and TCCSUP) were cultured in DMEM supplemented packaging vector pCL-Eco-DTM that lacked the transmembrane with 10% heat-inactivated fetal bovine serum, 2 mML- subunit of the envelope to generate pCL-EcoscFv. The p35 and p40 glutamine, and 50 mg/ml gentamicin. The cell cultures subunits of IL-12 as well as the sequence of the IRES were cloned were maintained at 37 1C in a humidified atmosphere with into retroviral expression vector pCLNRX to yield pCL-IL12. The pCL-LacZ and pCL-Eco plasmids served as the controls. pCLMFG- 5% CO2. Female C3H/HeN mice at the age of 6–8 weeks LacZ, pCLNRX, and pCL-Eco were from the Moloney murine were obtained from the Laboratory Animal Center of leukemia virus (MoMLV)-based pCL Vector System (RetroMax National Cheng Kung University Medical College and System, Imgenex). CMV, cytomegalovirus immediate-early promo- maintained under isothermal conditions with regular ter; RU5, direct repeat and unique sequence at 50 region of MoMLV photo periods. All the animal studies were performed RNA; c, packaging signal; LacZ, Escherichia coli b-galactosidase following the guidelines approved by the Laboratory gene; LTR, long terminal repeat of MoMLV; NeoR, neomycin Animal Care and Use Committee of the National Cheng phosphotransferase gene; RSV, Rous sarcoma virus promoter; Kung University. IL12p40, IL-12 p40 cDNA; IRES, encephalomyocarditis virus (EMCV) internal ribosome entry site; IL12p35, IL-12 p35 cDNA; SU, surface glycoprotein; TM, transmembrane; GAG, gag gene; Plasmid construction POL, pol gene; SP, signal peptide; RBD, receptor-binding domain;

The pCL-Eco-DTM plasmid was derived from the PRR, proline-rich region; C, carboxyl terminal domain; VH, cDNA for original pCL-Eco plasmid (Imgenex, San Diego, CA) in variable fragment of heavy chain; VL, cDNA for variable fragment of which 1-bp deletion at the 411-amino-acid site of the light chain.

Cancer Gene Therapy Enhanced killing of HER2-positive tumors by gammaretrovirus Y-S Tsai et al 39 IL-12 p40, as well as 50-GTCTTCTTGACGAGCATTC density was immediately determined by using an ELISA C-30 (forward) and 50-ACCTTCTGGGCATCCTTCA-30 reader set to 450 nm, with a wavelength correction at (reverse) for IRES. The PCR products were individually 570 nm. cloned into the pGEM-T easy vector (Promega, Madison, WI), and then transferred into the pCLNRX expression Cytotoxicity assay vector (Imgenex), yielding pCL-IL12 plasmid (Figure 1). Cells were plated at a density of 1 103 per well in 96-well The transcripts of anti-erbB2scFV and IL-12 were plates for overnight culture and infected with gammare- detected in 293 cells cotransfected with pCL-EcoscFv troviruses carrying the LacZ gene (Eco-LacZ or EcoscFv- and pCL-IL12 plasmids by RT-PCR (data not shown), LacZ) at a multiplicity of infection (MOI) of 1. When the verifying the correctness of the constructs. control wells were near 80–90% confluence, cell viability using an MTT assay was performed. MTT solution Generation of recombinant gammaretroviruses (1 mg/ml, 100 ml) was added to each well for 4 h at 37 1Cto To generate recombinant gammaretroviruses, 20 mg of the allow MTT to form formazan crystals by reacting with packaging vector and 20 mg of the expression vector were metabolically active cells. After the incubation was cotransfected into the 293 cells with the calcium completed, the supernatants were removed. Dimethyl phosphate precipitation method, according to the manu- sulfoxide (100 ml) was added, and transferred to another 26 facturer’s protocol. These viruses were designated as 96-well plate. The optical density of each well was Eco-LacZ, EcoscFv-LacZ, Eco-IL12, and EcoscFv-IL12, determined using an ELISA reader set to 570 nm. The respectively. The virus-containing supernatants were percentage of cell growth was calculated by comparing the harvested, filtered through 0.45-mm filters, and used absorbance reading from the treated vs the control cells. directly for various experiments or stored at 80 1C until needed. Cell cycle analysis MBT2 cells were seeded in 10-cm plates at a density of Determination of viral titers 5 1 10 per plate and infected with gammaretroviruses as NIH3T3 cells were plated at a density of 1 104 per well indicated at an MOI of 0.25 or 1. After 2 days, cells were in 24-well plates for overnight culture and were infected detached, washed with PBS, and resuspended in PI with four 10-fold dilutions of stored virus-containing staining buffer (50 mg/ml propidium iodide, 0.1% NP- supernatants in the presence of 8 mg/ml of polybrene for at 40, 10 mg/ml RNase A). After 30 min of incubation at least 12 h, following the manufacturer’s instruction. For room temperature, cell cycle analysis was performed on a recombinant gammaretroviruses that carried the b-galac- FACS Calibur Flow Cytometer (Becton Dickinson, San tosidase gene, the cells were stained with X-gal and the Jose, CA). The percentage of cells in each phase of the cell positive blue color-stained cells were calculated for cycle (G1, S, and G2/M) was calculated by using Modfit determination of the viral titer 48 h after viral infection. software (Becton Dickinson), and compared with the As for gammaretroviruses carrying the IL-12 gene, G418- results obtained with control untreated cells. resistant colonies (750 mg/ml) of infected NIH3T3 cells were counted and expressed as colony-forming units (CFU) per milliliter, as described earlier.27 Detection of apoptosis To test apoptotic death, MBT2 cells were seeded in 10-cm 5 Detection of gp70-anti-erbB2scFv chimera protein plates at a density of 1 10 per plate in the absence or To detect the presence of gp70-anti-erbB2scFv chimera presence of recombinant gammaretroviruses, as indicated protein in the viral supernatants, a modified enzyme- (each at an MOI of 1). After 2 days, cells were harvested, linked immunosorbent assay (ELISA) was performed. In washed in PBS, and treated with Annexin V-FITC after brief, diluted mouse ascites (1:180) containing monoclo- resuspension in binding buffer according to the manu- nal antibody against MoMLV envelope gp70 (ATCC, facturer’s instruction (Annexin V-FITC Apoptosis Detec- CRL-1913) or p15E (ATCC, CRL-1893) protein, or tion kit, Strong Biotech. Corp., Taipei, Taiwan). Labeled against prothymosin a (1D10) serving as a negative cells were analyzed using the FACS Calibur flow control was immobilized on 96-well ELISA plates at 4 1C cytometer (Becton Dickinson); the data were processed overnight. The plates were then washed three times with using Cell Quest software (Becton Dickinson). washing buffer (0.05% Tween-20/PBS) and blocked with blocking buffer (1% BSA/PBS) for 2 h. The 100 ml per Measurement of IL-12 and IFN-g by ELISA well of the viral supernatant were incubated overnight at The levels of IL-12 and IFN-g were determined by 4 1C. After washing, the plate was subsequently incubated commercial ELISA kits (R&D Systems) according to the for 2 h with recombinant HER2-Fc chimera protein manufacturer’s instruction. The biological activity of IL- (extracellular domain, 1129-ER, R&D Systems, Minnea- 12 was determined by the induction of IFN-g production. polis, MN) at a concentration of 2 mg/ml at room In brief, mouse splenocytes were harvested from three temperature, followed by addition of biotin-conjugated C3H/HeN mice, and 2 106 splenocytes were seeded in goat anti-human Fc (1:5000, R&D Systems) for another 24-well plates with Con A (2 mg/ml). IL-12-containing 2 h and streptavidin-peroxidase (KPL, Gaithersburg, supernatants as indicated were added. After 72 h, super- MD) for 20 min, and then reacted with 3,30,5,50-tetra- natants were collected for the measurement of IFN-g by methylbenzidine (Sigma, St Louis, MO). The optical ELISA.

Cancer Gene Therapy Enhanced killing of HER2-positive tumors by gammaretrovirus Y-S Tsai et al 40 Animal studies Results 6 MBT2 cells (2 10 cells in 100 ml PBS) were injected Determination of viral titers subcutaneously into the flanks of 6- to 8-week-old C3H/ NIH3T3 cells, 1 105 cells per well seeded in a 24-well HeN mice on day 0. On day 8, when visible and palpable plate overnight, were infected with serial 10-fold dilutions nodules developed, the mice with 10–11 mice per group of Eco-LacZ or EcoscFv-LacZ gammaretroviruses. X-gal were injected intratumorally with various recombinant 3 staining was done and the positive blue color-stained cells gammaretroviruses (5 10 CFU/dose) or with control were counted. The viral titers were about 1.1 105 CFU/ medium for 8 consecutive days. Tumor volume and ml for envelope-unmodified gammaretroviruses (Eco- mouse survival were recorded every 2 days according to LacZ) and 1.7 104 CFU/ml for envelope-modified the ‘Guidelines for the Welfare of Animals in Experi- gammaretroviruses (EcoscFv-LacZ). mental Neoplasia’ (1998) during the 60-day experimental period. Tumor volumes were calculated using the 2 Detection of gp70-anti-erbB2scFv chimera protein formula: (length of tumor) (width of tumor) 0.45. Using a modified ELISA with anti-gp70 as the capture The mean tumor volume was calculated while all the mice antibody, we showed that envelope-modified viral super- in the same treatment group were still alive. natant contained gp70-anti-erbB2scFv chimera proteins, as revealed by higher absorbance compared with unmodified viral supernatant or the control soluble anti- Determination of apoptotic cell number erbB2 scFv protein (Figure 2). When monoclonal anti- Typical apoptotic cell death in tumors, presenting as cell body against p15E (a TM subunit of the envelope) or shrinkage, with condensed hyperchromatic nuclear chro- prothymosin a (an unrelated protein serving as a negative matin and deeply eosinophilic cytoplasm surrounded by a control) was used as the capture antibody in the assay, all clear ‘halo’, occurs in a well-vascularized area full of samples had negligible absorbance. Furthermore, when proliferating tumor cells, some of which appear as mitotic no capture antibody was applied to the plates, all the figures. Tumor specimens obtained from three tumor- samples bound to the solid phase with various degrees, as bearing mice 5 days after five viral injections were revealed by different absorbance. Taken together, these harvested and embedded for hematoxylin and eosin results indicate that the modified ELISA using anti-gp70 staining. Microscopically, the number of apoptotic cells was calculated and averaged from every five high-power fields.

Assessment of intratumoral levels of IL-12, IFN-g, IL-1b, and tumor necrosis factor-a For detection of intratumoral levels of cytokines, MBT2 tumor-bearing mice that had been treated with various recombinant gammaretroviruses as described above for 5 consecutive days starting on day 8 were killed on day 17. A portion of tumor specimens were obtained and used to prepare total tumor cell lysates for the determination of levels of IL-12, IFN-g, IL-1b, and tumor necrosis factor (TNF)-a proteins using ELISA kits (R&D Systems) according to the manufacturer’s instruction. Data were presented after normalization with the tumor protein level. Figure 2 Detection of gp70-anti-erbB2scFv chimera protein in the EcoscFv-LacZ supernatant by a modified ELISA. The capture antibody included anti-gp70, anti-p15E, or anti-prothymosin a Statistical analysis (PTMA). The tested samples included Eco-LacZ and EcoscFv-LacZ Experimental results were analyzed for statistical signifi- viral supernatants as well as soluble anti-erbB2 scFv protein. cance (Student’s t-test) or curve-fit analysis using Prism Detection was based on biotin-conjugated goat anti-human Fc, GraphPad Prism 4.00 for Windows (GraphPad Software, followed by streptavidin-peroxidase and 3,30,5,50-tetramethylbenzi- San Diego, CA) and SigmaPlot 8.0 (SPSS Inc., Chicago, dine as the substrate. Note that envelope-modified viral supernatant IL) software packages. The difference in the mean relative contained gp70-anti-erbB2scFv chimera proteins, as revealed by tumor size±s.e.m. was examined using analyses of higher absorbance compared with unmodified viral supernatant or the control soluble anti-erbB2 scFv protein. When the capture covariance with days as the covariate using SPSS software antibody was not used in the assay, all the three samples bound to (SPSS 12.0 for Windows, SPSS Inc.). Results presented the solid phase with different degrees. Each value represents the are representative of at least duplicate experiments, and mean±s.e. (n ¼ 4; Eco-LacZ vs EcoscFv-LacZ; Po0.01 when the statistical significance is indicated (*Po0.05; **Po0.01; capture antibody was anti-gp70 or without capture antibody; ***Po0.001). Student’s t-test). **Po0.01; ***Po0.001.

Cancer Gene Therapy Enhanced killing of HER2-positive tumors by gammaretrovirus Y-S Tsai et al 41 as the capture antibody was specific for detection of gp70-anti-erbB2scFV and the EcoscFv-LacZ viral supernatant contained gp70-anti-erbB2ScFv chimera protein.

Infectivity and killing of HER2-expressing human bladder cancer cells To assess the infectivity and cytotoxic activity of the envelope-unmodified and -modified recombinant gammaretroviruses (Eco-LacZ and EcoscFv-LacZ), we infected several HER2-expressing bladder cancer cells for at least 12 h at an MOI of 1. The relative infectivity results showed that the envelope-modified gammaretroviruses EcoscFv-LacZ had a 10- to 1000-fold greater increase than the envelope-unmodified gammaretroviruses Eco-LacZ in HER2-expressing bladder cancer cells (Figures 3a and b). As for the effect on cell survival, EcoscFv-LacZ gammaretrovirus infection caused a 5–14% death rate among HER2-expressing tumor cells 2 days after infection. In contrast, in Eco-LacZ gammaretrovirus infections, about 98–100% of HER2-expressing tumor cells survived (Figure 3c).

Induction of apoptosis of MBT2 cells infected with EcoscFv-LacZ gammaretrovirus To determine whether the mechanism of cell death occurred after infection with EcoscFv-LacZ, we used annexin V to measure the appearance of phosphatidylserine, a marker of apoptosis, on the outer leaflet of the plasma membrane of MBT2 cells. Cells treated with EcoscFv-LacZ gammaretroviruses were found to bind more FITC-conjugated annexin V than untreated cells or cells treated with Eco-LacZ gammaretroviruses (Figure 4a). These results showed that the nature of cell death in EcoscFv-LacZ infection was apoptotic.

Effects of EcoscFv-LacZ gammaretroviruses on cell cycle of MBT2 cells To test whether a growth arrest contributed to the Figure 3 The relative infectivity and cell cytotoxicity of envelope- antitumor action of EcoscFv-LacZ, MBT2 cells were unmodified and -modified gammaretroviruses at an MOI of 1. (a and infected with Eco-LacZ or EcoscFv-LacZ for at least 12 h b) The results of relative infectivity showed that the envelope- at 37 1C. Two days later, cell cycle analysis by flow modified gammaretroviruses EcoscFv-LacZ have a 10–1000-fold cytometry was performed. EcoscFv-LacZ significantly greater increase than the envelope-unmodified gammaretroviruses reduced the percentage of MBT2 cells entering the G2/M Eco-LacZ in HER2-expressing bladder cancer cells. Each value represents the mean±s.d. (n ¼ 3; all Po0.0001 for Eco-LacZ vs phase and produced an accumulation of cells in the EcoscFv-lacZ; Student’s t-test). (c) Infection of EcoscFv-LacZ G0/G1 phase (Figure 4b). gammaretroviruses caused 5–14% of HER2-expressing tumor cell deaths 2 days after infection. In contrast, with the infection of Eco- Detection of IL-12 protein secreted from cells infected LacZ gammaretroviruses, about 98–100% of HER2-expressing with recombinant gammaretroviruses carrying the IL-12 tumor cells survived. Each value represents the mean±s.d. (n ¼ 3; gene Eco-LacZ vs EcoscFv-lacZ; Po0.05 for MBT2, TCCSUP, and T24 To demonstrate the expression and secretion of IL-12 cells; Po0.01 for J82 cells; Po0.001 for TSGH8301; Student’s protein in NIH3T3 cells infected with recombinant t-test). *Po0.05; **Po0.01; ***Po0.001. gammaretroviruses carrying the IL-12 gene, the supernatants from virus-producing 293 cells, G418-resistant NIH3H3 cells infected with Eco-IL12 or EcoscFv-IL12, as well as NIH3H3 cells infected with Eco-LacZ or cells, but the IL-12 amount was undetectable in the EcoscFv-LacZ were obtained and assessed for their IL-12 others. The amount of IL-12 in the supernatant collected contents by ELISA. As expected, the amount of IL-12 from G418-resistant NIH3T3 cells that had been infected was B1900 pg/ml in the supernatants from pCL-IL12- with Eco-IL12 or EcoscFv-IL12 was B2200 pg/ml transfected, Eco-IL12- and EcoscFv-IL12-producing 293 (Figure 5a).

Cancer Gene Therapy Enhanced killing of HER2-positive tumors by gammaretrovirus Y-S Tsai et al 42

Cancer Gene Therapy Enhanced killing of HER2-positive tumors by gammaretrovirus Y-S Tsai et al 43 Induction of IFN-g production from Con A-primed significantly higher in the tumors treated with EcoscFv- splenocytes IL12 than those in the remaining four groups (Figures 8c To determine whether the secreted IL-12 protein shown in and d). Figure 5a exhibits biological activity, we performed an IFN-g induction assay. As shown in Figure 5b, addition of recombinant IL-12 enhanced the production of IFN-g by Con A-primed splenocytes. Increased IFN-g produc- Discussion tion was also noted in the supernatant of Con A-primed In this study, we altered the tropism of the gammare- splenocytes in the presence of the conditioned medium troviral vector to infect HER2-expressing human bladder obtained from EcoscFv-IL12-infected NIH3T3 cells, as cancer cells through the recognition of the HER2 compared with that from EcoscFv-LacZ-infected cells molecule on the tumor cell surface, and the IL-12 protein (Figure 5b). can be expressed after infection. In contrast with wild- type MoMLV, the anti-erbB2scFv-modified MoMLV In vivo antitumor activity carrying reporter genes can extend their tropism to To investigate the in vivo antitumor activity of envelope- several HER2-expressing human bladder cancer cell lines. unmodified or -modified gammaretroviruses carrying Moreover, such gammaretroviruses can induce tumor cell LacZ or the IL-12 gene, we compared MBT2 tumor- apoptosis and influence the cell cycle. With a synergistic bearing mice treated intratumorally with each viral effect from IL-12 protein and the binding process of the supernatant, including control medium, Eco-LacZ, HER2 receptor-anti-erbB2scFv minibody, the antitumor EcoscFv-LacZ, Eco-IL12, and EcoscFv-IL12. As shown activity was enhanced and the survival of the mice was in Figures 6a and b, EcoscFv-LacZ or Eco-IL12 treat- prolonged, which were associated with increased apopto- ment exerted limited antitumor activity as compared with sis, IL-12, IFN-g, IL-1b, and TNF-a within the tumors. Eco-LacZ or vehicle control treatment. However, treat- The host range of gammaretroviruses is dependent on ment with EcoscFv-IL12 exerted higher antitumor the SU, which recognizes and binds to a specific cell activity and prolonged the survival of tumor-bearing receptor protein. The ecotropic MoMLV recognizes mice compared with Eco-IL12 or vehicle control treat- receptors only on rodent cells, and rarely on human cells, ment. Taken together, the antitumor activity of gammar- and its host range is restricted to rodent cells.26 One of the etroviruses carrying the IL-12 gene was enhanced through challenges in the engineering of gammaretroviral envelope genetic modification of the envelope targeting HER2 proteins is to direct viruses to cell surface molecules receptor. expressed by tumor cells without impairing viral entrance. Several key steps have to be overcome: the modified Increased number of apoptotic cells envelope has to be functionally expressed in the packaging After staining and counting, there was significantly more cells, the envelope has to become incorporated into the apoptotic cells in tumors treated with envelope-modified viral particles, the viral particles have to attach to the cell gammaretroviruses (EcoscFv-LacZ and EcoscFv-IL12) of choice, membrane fusion has to be triggered, and the than those treated with unmodified ones (Eco-LacZ and gene of interest has to be expressed. Previous studies have Eco-IL12) or the control (Figures 7a and b). shown that the generation of gammaretroviral vectors incorporated with a scFv antibody fused to envelope Increased cytokine levels in tumors treated with protein could be inserted close to the N terminus of the EcoscFv-IL12 gammaretrovirus ecotropic MoMLV envelope protein,15 or to the PRR of The results from the ELISA showed that IL-12 levels were the surface glycoprotein that has been used to insert higher in tumors treated with Eco-IL12 or EcoscFv-IL12, molecules of interest on amphotropic, but not ecotropic but were undetectable in those treated with Eco-LacZ or MoMLV.14,28 Generally, the success of infection should the control. A relatively lower IL-12 level could be be aided by the presence of wide-type envelope (un- detected within the tumor treated with EcoscFv-LacZ modified) protein; the ratio of wide-type and modified (Figure 8a). Similarly, the levels of IFN-g were signifi- envelope protein in the viral particles appears to cantly higher in the tumors treated with Eco-IL12 or determine the efficiency of infection. The presence of EcoscFv-IL12 than those treated with Eco-LacZ, large amounts of wide-type envelopes may inhibit binding EcoscFv-LacZ, or the control (Figure 8b). On the other of the modified envelope to its antigen on the cell surface. hand, the levels of both IL-1b and TNF-a were The existence of a modified envelope alone has been

Figure 4 Induction of apoptosis and effect of cell cycle progression by envelope-modified recombinant gammaretrovirus (EcoscFv-LacZ). (a) MBT2 cells were infected with envelope-unmodified or -modified gammaretroviruses at an MOI of 1. Two days later, the amount of annexin V staining was analyzed with flow cytometry. The result showed EcoscFv-LacZ-infected MBT2 cells exhibited a higher MFI as compared with those infected with Eco-LacZ or incubated with a control medium. Each value represents the mean±s.d. (n ¼ 3; Ecoscfv-LacZ vs Eco-LacZ or control; Po0.05, Student’s t-test). (b) MBT2 cells were infected as indicated. Two days later, analysis of the cell cycle with propidium iodide staining showed that treatment of envelope-modified EcoscFv-LacZ influenced cell cycle progression. Each value represents the mean±s.d. (n ¼ 3; G2/M phase, Ecoscfv-LacZ vs Eco-LacZ or control; Po0.05, Student’s t-test). *Po0.05.

Cancer Gene Therapy Enhanced killing of HER2-positive tumors by gammaretrovirus Y-S Tsai et al 44

Figure 6 In vivo inhibition of tumor growth by EcoscFv-IL12. MBT2 tumor-bearing mice were treated intratumorally with various recombinant gammaretroviruses (5 103 CFU/dose) or with control supernatants (CM) from single expression vector-transfected 293 cells for 8 consecutive days starting on day 8. (a) Mean tumor volumes and (b) Kaplan–Meier survival curves. Note that EcoscFv- IL12 treatment significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice (n ¼ 10, control, Eco-LacZ & Eco-IL groups; n ¼ 11, EcoscFv-LacZ group; n ¼ 12, EcoscFv-IL12 group; Po0.05 for EcoscFv-IL12 vs Eco-IL12, Eco-IL12 vs Eco-LacZ, and Figure 5 Detection of IL-12 protein in NIH3T3 cells infected with EcoscFv-LacZ vs Eco-LacZ; Po0.01 for EcoscFv-IL12 vs EcoscFv- Eco-IL12 or EcoscFv-IL12 gammaretroviruses and their biological LacZ, Eco-LacZ, or control; log-rank test). *Po0.05; **Po0.01; activities. (a) Culture supernatants were obtained from virus- ***Po0.001. producing 293 cells after cotransfection of plasmids (pCL-LacZ alone, pCL-LacZ/pCL-Eco, pCL-LacZ/pCL-EcoscFv, pCL-IL12 suggested to have too much affinity to initiate membrane alone, pCL-IL12/pCL-Eco, pCL-IL12/pCL-EcoscFv), from G418- fusion after conformational changes of receptor bind- resistant, Eco-IL12- or EcoscFv-IL12-infected NIH3T3 cells, or from ing.29 For example, amphotropic gammaretroviral envel- Eco-LacZ- or EcoscFv-LacZ-infected NIH3T3 cells, and their IL-12 contents were determined by ELISA. Each column represents values ope glycoproteins that display EGF cannot infect human of three measurements, and the mean value is indicated by cells that express both EGF receptors and amphotropic 30,31 horizontal bar (n ¼ 3). (b) Induction of IFN-g production. Culture receptors. Inhibition of infectivity is specifically supernatants, which were collected from NIH3T3 cells infected with caused by interaction with the high affinity EGF receptor, EcoscFv-LacZ or EcoscFv-IL12, were incubated with Con A-primed as competition with soluble EGF can restore infectivity mouse splenocytes. After 3 days, the amount of IFN-g protein was through the amphotropic receptor. Since the TM protein, determined by ELISA. Each value represents the mean±s.d. (n ¼ 3; but not the SU of the gammaretroviral envelope, is Po0.05 for control medium vs recombinant IL-12-containing required for envelope fusion,32 some mutations located medium; Po0.01 for NIH-LacZ vs NIH-IL12 group; Student’s t-test). within the TM region (TM-T25P, TM-L57V) and the Con A-free conditioned medium served as a negative control. CM, presence of the R peptide of the TM subunit contributes culture medium. *P 0.05; **P 0.01. o o to the inhibition of envelope fusion. In this study, despite the lack of TM protein, anti-erbB2scFv,

Cancer Gene Therapy Enhanced killing of HER2-positive tumors by gammaretrovirus Y-S Tsai et al 45

Figure 7 Numbers of apoptotic cells within the tumors. (a) H&E staining ( 320) revealed that numbers of apoptotic cells in proliferative tumors treated with envelope-modified viruses (EcoscFv-LacZ and EcoscFv-IL12) were significantly greater than those treated with envelope-unmodified ones (Eco-LacZ and Eco-IL12) and the control medium (CM). A and M are apoptotic and mitotic cells, respectively. V denotes blood vessels. Lower right, magnified image ( 640 magnification) of the area indicated by the box (lower middle) from EcoScFV-IL12-treated tumor. (b) Each value represents the mean±s.e. (n ¼ 3; Po0.05 for EcoscFv-IL12 vs Eco-IL12; Po0.01 for EcoscFv-LacZ vs Eco-LacZ or control, Student’s t-test). *Po0.05; **Po0.01. envelope-modified gammaretroviruses are still capable of rapid than the MTT assay in the aspect of cell death. On infecting target cells and expressing reporter gene and IL- the other hand, although the methods of purification and 12 protein, suggesting that the entry of the gene of interest concentration with polybrene and chrondoitin sulfate C may rely on erbB2-mediated endocytosis through the were reported to be useful for amphotropic and ecotropic binding of the HER2 receptor and anti-erbB2scFv. recombinant retroviruses,33 no report discussed whether In this study, only a 5–14% death rate of infected cells the concentration methods work well for envelope- found in the MTT assay 2 days after viral infection may modified recombinant gammaretroviruses that lack TM reflect lower viral titers. The inconsistencies between the domain. As we were concerned that the concentration results from the MTT assay and annexin V staining may procedures may change the surface configuration of result from the time process during the cell death. The scFv,34 resulting in affecting the antigen-binding ability, former measures the mitochondrial activity during cell we did not use this procedure in this study. death, whereas the latter detects the appearance of In our studies, the anti-erbB2scFv-modified MMLV phosphatidylserine in the initial phase of cell apoptosis. could infect HER2-expressing cancer cells without com- Therefore, detection of annexin V is more sensitive and bining wild-type envelope. We also demonstrated that

Cancer Gene Therapy Enhanced killing of HER2-positive tumors by gammaretrovirus Y-S Tsai et al 46 a combination of T and NK cell activity, a switch to a Th1 immune response, and antiangiogenic activity.38 The results from the current animal studies showed that treatment with envelope-modified gammaretrovirus targeting the HER2 receptor carrying the LacZ gene exerts a weak antitumor activity. When carrying the IL-12 gene, the efficacy of antitumor activity was enhanced, initially suggesting the synergistic effect derived from the binding of the HER2 antigen and anti-erbB2scFv and IL-12 protein. Further analyses from the tumor specimen demonstrated that therapies with envelope-modified gammaretroviruses can induce more tumor cell apoptosis. As apoptotic cell debris may be removed and recognized by macrophage, it may cause macrophage phenotype shift and decrease the production of TNF-a and IL-12 proteins.39 Despite this, it was reported that exogenous IL-12 can potently activate macrophages during intracellular infection, such as mycobacteria, and this activating Figure 8 Intratumoral levels of IL-12, IFN-g, IL-1b, and TNF-a. effect through TNF-a release is mediated primarily MBT2 tumor-bearing mice were treated intratumorally with various 3 through its effect on the release of IFN-g by macro- recombinant gammaretroviruses (5 10 CFU/dose) or with control phages.40 Like this, such an antitumor activity in this medium (CM) for 5 consecutive days starting on day 8. On day 17, mice were killed and their tumors were excised for preparing tumor study might be enhanced through IL-12 and IL-12- cell lysates. Intratumoral levels of cytokines were determined by mediated IFN-g production. ELISA. (a) Higher levels of intratumoral IL-12 protein were found in In conclusion, our results show that production of anti- tumors treated with Eco-IL12 or EcoscFv-IL12, whereas it was erbB2scFv-modified MoMLV is independent of the wild- undetectable in those treated with Eco-LacZ or control medium. A type envelope combination. In addition, the modified relatively lower level of IL-12 protein could be detected within the gammaretroviruses exerted antitumor activity through tumors treated with EcoscFv-LacZ. (b) The levels of intratumoral targeting and binding of anti-erbB2scFv to HER2 IFN-g protein were significantly higher in the tumors treated with Eco- receptor on bladder cancer cells. When this modified IL12 or EcoscFv-IL12 than those treated with Eco-LacZ, EcoscFv- gammaretrovirus expresses the IL-12 gene, it demon- LacZ, or control medium (n ¼ 3; Po0.05 for Eco-IL12 or EcoscFv- strates a better therapeutic efficacy compared with that of IL12 vs Eco-LacZ, EcoscFv-LacZ, or control, Student’s t-test). (c and d) The levels of intratumoral IL-1b and TNF-a proteins were the unmodified one, which is associated with increased significantly higher in the tumors treated with EcoscFv-IL12 than production of TNF-a and IL-1b. Such an anti-erbB2scFv- those in the remaining four groups (n ¼ 3; Po0.05 for EcoscFv-IL12 modified MoMLV carrying the IL-12 gene may be a vs Eco-IL12, Eco-LacZ, EcoscFv-LacZ, or control, Student’s t-test). promising strategy for HER2-positive bladder cancer Each value represents the mean±s.e. *Po0.05; ***Po0.001. therapy.

modified MoMLV infected tumor cells by means of anti- erbB2scFv fused protein and signal transduction leading Conflict of interest to cell death. Recently, these minibodies have been The authors declare no conflict of interest. demonstrated to exhibit unique functional properties. They not only induce internalization, but also display an intrinsic, strong, and specific anti-proliferative activity Acknowledgements toward HER2-positive cells.11 Moreover, a new anti- erbB2 antibody, Erb-hcAb, by fusing with a human anti- This study was supported by the National Science Council erbB2scFv selectively cytotoxic to erbB2-positive cells, of Taiwan (Grants NSC-94-2314-B-006-090, NSC-93- and a human Fc domain, can induce apoptosis and inhibit 2314-006-070, NSC-92-2314-B-006-135, and NSC-91- cell cycle progression in erbB2-positive cells.35 However, 2314-B-006-148) and Hospital of Department of Health, as shown in this study, cell death caused by modified Executive Yuan, Taiwan (Grants 93033 and 9413). gammaretroviruses was independent of HER2 expression, suggesting some HER2-expressing cells may or may not be dependent on p185-mediated mitogenic signaling and References have some cross-talk signaling.36 Several studies showed that IL-12 is an important 1 Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, cytokine for ex vivo recovery and maintenance of HER2 Keith DE et al. Studies of the HER-2/neu proto-oncogene antigen-specific CD4 þ T lymphocytes that might be lost 24,37 in human breast and ovarian cancer. Science 1989; 244: by using IL-2 alone in combination with antigen. 707–712. Moreover, an anti-erbB2 antibody-IL-12 chimeric protein 2 Hussain MH, MacVicar GR, Petrylak DP, Dunn RL, exerts an antitumor activity, and the mechanism involves Vaishampayan U, Lara Jr PN et al. Trastuzumab, paclitaxel,

Cancer Gene Therapy Enhanced killing of HER2-positive tumors by gammaretrovirus Y-S Tsai et al 47 carboplatin, and gemcitabine in advanced human epidermal cells by IL-12 is dependent on IFN-gamma and VCAM-1 growth factor receptor-2/neu-positive urothelial carcinoma: and is rapidly down-regulated by a mechanism involving T results of a multicenter phase II national cancer institute cells and expression of Fas. J Immunol 1998; 161: 6014–6021. trial. J Clin Oncol 2007; 25: 2218–2224. 18 Nastala CL, Edington HD, McKinney TG, Tahara H, 3 Kru¨ger S, Weitsch G, Bu¨ttner H, Matthiensen A, Bo¨hmer T, Nalesnik MA, Brunda MJ et al. Recombinant IL-12 Marquardt T et al. HER2 overexpression in muscle-invasive administration induces tumor regression in association with urothelial carcinoma of the bladder: prognostic implications. IFN-gamma production. J Immunol 1994; 153: 1697–1706. Int J Cancer 2002; 102: 514–518. 19 Brunda MJ, Luistro L, Warrier RR, Wright RB, Hubbard 4 Tsai YS, Tzai TS, Chow NH, Yang WH, Tong YC, Lin JS BR, Murphy M et al. Antitumor and antimetastatic activity et al. Prognostic values of p53 and HER-2/neu coexpression of interleukin 12 against murine tumors. J Exp Med 1993; in invasive bladder cancer in Taiwan. Urol Int 2003; 71: 178: 1223–1230. 262–270. 20 Voest EE, Kenyon BM, O’Reilly MS, Truitt G, D’Amato 5 Lonn U, Lonn S, Friberg S, Nilsson B, Silfversward C, RJ, Folkman J. Inhibition of angiogenesis in vivo by Stenkvist B. Prognostic value of amplification of interleukin 12. J Natl Cancer Inst 1995; 87: 581–586. c-erb-B2 in bladder carcinoma. Clin Cancer Res 1995; 1: 21 Shi X, Cao S, Mitsuhashi M, Xiang Z, Ma X. Genome-wide 1189–1194. analysis of molecular changes in IL-12-induced control of 6 Gandour-Edwards R, Lara Jr PN, Folkins AK, LaSalle JM, mammary carcinoma via IFN-gamma-independent mechan- Beckett L, Li Y et al. Does HER2/neu expression provide isms. J Immunol 2004; 172: 4111–4122. prognostic information in patients with advanced urothelial 22 Rodolfo M, Colombo MP. Interleukin-12 as an adjuvant for carcinoma? Cancer 2002; 95: 1009–1015. cancer immunotherapy. Methods 1999; 19: 114–120. 7 Shiau AL, Lin YP, Shieh GS, Su CH, Wu WL, Tsai YS et al. 23 Portielje JE, Gratama JW, van Ojik HH, Stoter G, Kruit Development of a conditionally replicating pseudorabies WH. IL-12: a promising adjuvant for cancer vaccination. virus for HER-2/neu-overexpressing bladder cancer therapy. Cancer Immunol Immunother 2003; 52: 133–144. Mol Ther 2007; 15: 131–138. 24 Chen Y, Emtage P, Zhu Q, Foley R, Muller W, Hitt M et al. 8 Lin CC, Chou CW, Shiau AL, Tu CF, Ko TM, Chen YL Induction of ErbB-2/neu-specific protective and therapeutic et al. Therapeutic HER2/Neu DNA vaccine inhibits mouse antitumor immunity using genetically modified dendritic tumor naturally overexpressing endogenous neu. Mol Ther cells: enhanced efficacy by cotransduction of gene encoding 2004; 10: 290–301. IL-12. Gene Ther 2001; 8: 316–323. 9 Suzuki M, Shinkai M, Honda H, Kamihira M, Iijima S, 25 Bramson JL, Hitt M, Addison CL, Muller WJ, Gauldie J, Kobayashi T. Construction of tumor-specific cells expressing Graham FL. Direct intratumoral injection of an adenovirus a membrane-anchored single-chain Fv of anti-erbB-2 anti- expressing interleukin-12 induces regression and long-lasting body. Biochim Biophys Acta 2001; 1525: 191–196. immunity that is associated with highly localized expression 10 Arafat WO, Go´mez-Navarro J, Buchsbaum DJ, Xiang J, of interleukin-12. Hum Gene Ther 1996; 7: 1995–2002. Wang M, Casado E et al. Effective single chain 26 Naviaux RK, Costanzi E, Haas M, Verma IM. The pCL antibody (scFv) concentrations in vivo via adenoviral vector vector system: rapid production of helper-free, high-titer, mediated expression of secretory scFv. Gene Ther 2002; 9: recombinant retroviruses. J Virol 1996; 70: 5701–5705. 256–262. 27 Shiau AL, Lin CY, Tzai TS, Wu CL. Postoperative immuno- 11 De Lorenzo C, Palmer DB, Piccoli R, Ritter MA, D’Alessio gene therapy of murine bladder tumor by in vivo adminis- G. A new human antitumor immunoreagent specific for tration of retroviruses expressing mouse interferon-gamma. ErbB2. Clin Cancer Res 2002; 8: 1710–1719. Cancer Gene Ther 2001; 8: 73–81. 12 Neve RM, Nielsen UB, Kirpotin DB, Poul M-A, Marks JD, 28 Tai CK, Logg CR, Park JM, Anderson WF, Press MF, Benz CC. Biological effects of anti-erbB2 single chain Kasahara N. Antibody-mediated targeting of replication- antibodies selected for internalizing function. Biochem competent retroviral vectors. Hum Gene Ther 2003; 14: Biophys Res Commun 2001; 280: 274–279. 789–802. 13 Lorimer IA, Lavictoire SJ. Targeting retrovirus to cancer 29 Chu TH, Dornburg R. Toward highly efficient cell-type- cells expressing a mutant EGF receptor by insertion of a specific gene transfer with retroviral vectors displaying single chain antibody variable domain in the envelope single-chain antibodies. J Virol 1997; 71: 720–725. glycoprotein receptor binding lobe. J Immunol Methods 30 Nilson BH, Morling FJ, Cosset FL, Russell SJ. Targeting of 2000; 237: 147–157. retroviral vectors through protease-substrate interactions. 14 Nguyen TH, Loux N, Dagher I, Vons C, Carey K, Briand P Gene Ther 1996; 3: 280–286. et al. Improved gene transfer selectivity to hepatocarcinoma 31 Cosset FL, Morling FJ, Takeuchi Y, Weiss RA, Collins MK, cells by retrovirus vector displaying single-chain variable Russell SJ. Retroviral retargeting by envelopes expressing an fragment antibody against c-Met. Cancer Gene Ther 2003; N-terminal binding domain. J Virol 1995; 69: 6314–6322. 10: 840–849. 32 Russell SJ, Cosset FL. Modifying the host range properties 15 Khare PD, Shao-Xi L, Kuroki M, Hirose Y, Arakawa F, of retroviral vectors. J Gene Med 1999; 1: 300–311. Nakamura K et al. Specifically targeted killing of carcinoem- 33 Landazuri N, Gupta M, Le Doux JM. Rapid concentration bryonic antigen (CEA)-expressing cells by a retroviral vector and purification of retrovirus by flocculation with polybrene. displaying single-chain variable fragmented antibody to J Biotechnol 2006; 125: 529–539. CEA and carrying the gene for inducible nitric oxide 34 Watkins NA, Dafforn TR, Kuijpers M, Brown C, Javid B, synthase. Cancer Res 2001; 61: 370–375. Lehner PJ et al. Molecular studies of anti-HLA-A2 using 16 Hunter CA. New IL-12-family members: IL-23 and IL-27, light-chain shuffling: a structural model for HLA antibody cytokines with divergent functions. Nat Rev Immunol 2005; 5: binding. Tissue Antigens 2004; 63: 345–354. 521–531. 35 De Lorenzo C, Cozzolino R, Carpentieri A, Pucci P, Laccetti 17 Fogler WE, Volker K, Watanabe M, Wigginton JM, P, D0Alessio G. Biological properties of a human compact Roessler P, Brunda MJ et al. Recruitment of hepatic NK anti-ErbB2 antibody. Carcinogenesis 2005; 26: 1890–1895.

Cancer Gene Therapy Enhanced killing of HER2-positive tumors by gammaretrovirus Y-S Tsai et al 48 36 Nahta R, Yu D, Hung MC, Hortobagyi GN, Esteva FJ. (mscIL-12.her2.IgG3). J Interferon Cytokine Res 2001; 21: Mechanisms of disease: understanding resistance to HER2- 709–720. targeted therapy in human breast cancer. Nat Clin Pract 39 Weigert A, Tzieply N, von Knethen A, Johann AM, Schmidt Oncol 2006; 3: 269–280. H, Geisslinger G et al. Tumor cell apoptosis polarizes 37 Knutson KL, Disis ML. IL-12 enhances the generation macrophages role of sphingosine-1-phosphate. Mol Biol Cell of tumour antigen-specific Th1 CD4 T cells during 2007; 18: 3810–3819. ex vivo expansion. Clin Exp Immunol 2004; 135: 40 Xing Z, Zganiacz A, Santosuosso M. Role of IL-12 in 322–329. macrophage activation during intracellular infection: IL-12 38 Peng LS, Penichet ML, Dela Cruz JS, Sampogna SL, and mycobacteria synergistically release TNF-alpha and Morrison SL. Mechanism of antitumor activity of a nitric oxide from macrophages via IFN-gamma induction. single-chain interleukin-12 IgG3 antibody fusion protein J Leukoc Biol 2000; 68: 897–902.

Cancer Gene Therapy