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1 Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee; 2 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Lung Institute of Medical Research Center, Seoul National University College of Medicine and Respiratory Center, Seoul National University Bundang Hospital, Seongnam, Korea; and 3 Gene Therapy Center, University of Alabama at Birmingham, Birmingham, Alabama
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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24RGD) that produces a mutant E1 without the ability to bind retinoblastoma but retaining viral replication competence in cancer cells with a defective pRb/p16. Ad-lacZ, adenovirus-luciferase (ad-luc), and adenovirus insulin-like growth factor-1R/dominant-negative (ad-IGF-1R/dn; 482, 950) are E1-deleted replication-defective adenoviruses. The combination of CRAD and ad-lacZ increased the transduction efficiency of lacZ to 100% from 15% observed with ad-lacZ alone. Transfer of media of CRAD and ad-lacZ cotransduced cells induced the transfer of lacZ (media transferable bystander effect). Combination of CRAD and ad-IGF-1R/dn increased the production of truncated IGF-1R or soluble IGF-1R > 10 times compared with transduction with ad-IGF-1R/dn alone. Combined intratumoral injection of CRAD and ad-luc increased the luciferase expression about 70 times compared with ad-luc alone without substantial systemic spread. Combined intratumoral injection of CRAD and ad-IGF-1R/482 induced stronger growth suppression of established lung cancer xenografts than single injections. The combination of CRAD and E1-deleted ad induced tumor-specific replication of CRAD and E1-deleted ad and increased the transduction rate and therapeutic efficacy of these viruses in model tumors. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One strategy for overcoming this problem is the use of a conditionally replicating adenovirus (CRAD) that can selectively replicate in tumor cells (4 , 5) . The antitumor effect of most CRAD depends on the antitumor effects intrinsic to adenovirus replication in tumor cells and dissemination through tumor mass.
Comparative studies between CRAD and replication-defective viruses have shown CRAD to induce higher and more prolonged expression of transgenes (6) . However, clinical studies with CRAD failed to show striking results although some responses were observed (7 , 8) . Most of these viruses cannot carry a therapeutic gene because of size constraints. Furthermore, the genetic heterogeneity of tumors is the problem for this approach because typical CRAD can replicate only in p53-mutated (4 , 5 , 9) , pRB/p16 pathway-inactivated cells (10, 11, 12) , or cells expressing a specific protein (CRAD containing cancer-specific promoters such as the telomerase promoter; refs. 13 and 14 ).
Another strategy is the use of CRAD engineered to also express a therapeutic gene inserted into the E1 or E3 region of adenovirus (an "armed" therapeutic virus; refs. 15, 16, 17, 18, 19, 20, 21, 22 ). This strategy enhances the therapeutic effects of CRAD by coexpression of the therapeutic gene; however, the problem of low gene transfer rate is still limiting because the effect of the therapeutic gene is confined to the cells transduced with the CRAD.
Here we hypothesized that a conventional replication-defective adenovirus containing an E1 deletion can become replication-competent when cotransduced with a CRAD that would supply E1 in trans. The resulting selective production of large numbers of the therapeutic adenovirus in situ within a tumor mass could transduce neighboring tumor cells and increase the overall transduction efficiency.
The basic idea of this study has already been reported. Habib et al. (23) demonstrated that the combination of replication competent adenovirus with replication-defective adenovirus with reporter genes (green florescent protein and luciferase) induced the replication of reporter viruses.
In these studies, we used a CRAD designated 24RGD that produces a mutant E1 protein without the ability to bind retinoblastoma (Rb) but retaining viral replication competence. This E1 can theoretically only function and permit viral replication in cancer cells with a defective pRb/p16 pathway. Also, this 24RGD contains a Arg-Gly-Asp (RGD) sequence, known to interact with 
v integrins, in adenoviral fiber that facilitate adenoviral entry to cells regardless of Coxsackie adenoviral receptor (24)
.
For the replication-defective therapeutic adenovirus, we tested adenoviruses expressing a dominant-negative insulin-like growth factor-1R (ad-IGF-1R/dn) with a stop codon at codon 950 (ad-IGF-1R/950) or 482 (ad-IGF-1R/482) for the genetic blockade of IGF-1. IGF-1 is very important growth factor essential for the cancer growth. We have already demonstrated that these adenoviruses effectively blocked the IGF-1 pathway in cancer cells and that intratumoral injection of ad-IGF-1R/482 [1 x 109 plaque-forming unit(s) (pfu) x 5 times] suppresses growth of lung cancer xenografts (25) by effectively blocking IGF-1 signaling.
In this study, we showed that combined transduction with 24RGD and an ad-IGF-1R/dn increased the transduction efficiency and therapeutic efficacy of either approach alone on lung cancer xenografts.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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24RGD was constructed in the Gene Therapy Center at the University of Alabama at Birmingham. This virus contains E1A with a 24-bp deletion in the CR2 region and E3 with an RGD-4C modification of the fiber gene. The CR2 domain is responsible for binding pRb that allows the adenovirus-infected cell to enter S phase. Therefore adenoviruses with a deletion in this region can replicate only in the cells with defects in the pRb/p16 pathway where this binding is not necessary. Furthermore, the RGD-4C motif in the E3 region enables this virus to infect cells without binding to Coxsackie adenoviral receptor (24)
.
We have previously described the construction of adenoviruses expressing IGF-1R/dn, 950 amino acids, or 482 amino acids in length (25)
. Ad-IGF-1R/950 produced truncated IGF-1R receptor on the cell surface, and ad-IGF-1R/482 produced and secreted a defective subunit of IGF-1R. These ad-IGF-1R/950, 482, ad-lacZ, and adenovirus-luciferase (ad-luc) were driven by cytomegalovirus promoter and were replication defective by virtue of E1 deletion.
Increased In vitro Transduction Efficiency of ad-lacZ or ad-luc by Cotransduction with 24RGD.
To assess the change in transduction efficiency of ad-lacZ in combination with 24RGD in vitro, we transduced NCI H460 (human lung large cell carcinoma) with ad-lacZ [10 multiplicity of infection (moi)] alone or ad-lacZ (10 moi) and 24RGD (1 moi) together. Twenty-four hours after transduction, we measured the efficiencies of lacZ gene transfer by 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) staining. The same experiment was performed with ad-luc (5 moi) and 24RGD (1 moi). Twenty-four hours after the transduction, the expression of firefly luciferase were measured by luciferase assay with Luciferase Assay System (E4030, Promega, Madison, WI) from cell lysates with the Luciferase Cell Culture Lysis Reagent (Promega).
The Presence of Intact ad-lacZ in the Media of ad-lacZ- and 24RGD-Transduced Cells.
After combined transduction with ad-lacZ (10 moi) with or without 24RGD (1 moi) in NCI H460, we washed the cells with PBS to remove the viruses remaining in solution. Twenty-four hours after transduction, we collected and concentrated the media by Centriplus-30 (Millipore, Billerica, MA) and then transferred this medium to uninfected NCI H460 cells. Twenty-four hours after the transfer, we performed X-gal staining to confirm the release of ad-lacZ into medium by counting the blue cells expressing lacZ.
The Expression of Transgenes Encoded by ad-IGF-1R/950 and 482 after Combined Transduction with 24RGD.
Transduction with ad-IGF-1R/950 results in the production of a truncated IGF-1R on the cell surface. We thus measured and compared IGF-1R cell surface expression by fluorescence-activated cell sorter. Twenty-four hours after combined transduction with ad-IGF-1R/950 (10 moi) with or without 24RGD (2 moi) in NCI H460 cells, we measured the IGF-1R expression by flow cytometry with the IR3 antibody (Oncogene Research Products, Cambridge, MA). The same experiment was repeated with ad-IGF-1R/482 (10 moi) and 24RGD (2 moi). We measured IGF-1R protein by Western blot assay with an antibody for the subunit (anti-IGF-1R N-20, Santa Cruz Biotechnology, Santa Cruz, CA) from protein extracts of cells and media after transduction with ad-IGF-1R/482 and/or 24RGD because ad-IGF-1R/482 has been demonstrated to produce and release the truncated subunit of IGF-1R into the medium.
Increased Transduction Efficiency and Transgene Expression of lacZ or Luciferase in Tumors after Coinjection of ad-lacZ and 24RGD.
We established subcutaneous xenograft tumors by injecting NCI H460 (1 x 106 cells/mouse) into the flank of nude mice. Seven days after injection, we injected ad-lacZ (1 x 107 pfu) with or without 24RGD (1 x 107 pfu) into the palpable tumor mass. Seven days after adenoviral injection, we excised the tumor and stained with X-gal to measure expression of transduced lacZ activity.
With ad-luc, we performed a similar experiment except we induced bilateral subcutaneous tumors to evaluate both transduction efficiency and systemic spread of ad-luc to other organs and tumors at a distant site. Seven days after tumor cell injections into the subcutaneous tissue of both flanks, we injected ad-luc (1 x 107 pfu) with or without 24RGD (1 x 107 pfu) into one tumor directly, and 1 week later we excised both tumors (treated and untreated), liver, lung, spleen, and kidney. We homogenized these tissues, mixed the homogenate with Luciferase Cell Culture Lysis Reagent (Promega) followed by emulsification. After centrifugation, we transferred 20 µL of supernatant into 100 µL of Luciferase Assay Reagent in a luminometer tube and assayed it in a luminometer (Luciferase Assay System, Promega). We measured the protein concentration of the supernatant with the Bradford method and compared the transduction efficiency by normalizing for relative light units per microgram of protein across samples (26)
. The tumor mass and organs from untreated mice were used as background assays.
Growth Suppression after Combined Transduction with 24RGD and ad-IGF-1R/dn (482, 950).
NCI H460 cells were plated into 6-well plates (2 x 104 cells/well). Twenty-four hours later, they were transduction with 24RGD (0.1 moi), ad-IGF-1R/482 (1 moi), ad-IGF-1R/950 (1 moi), 24RGD (0.1 moi) + ad-IGF-1R/482 (1 moi), and 24RGD (0.1 moi) + ad-IGF-1R/950 (1 moi). Cell numbers were counted with a hemocytometer and compared with untransduced controls for relative survival.
Tumor Treatment.
To investigate the effect of combination therapy on the treatment of established tumors, we performed intratumoral injections of 24RGD and/or ad-IGF-1R/482 into subcutaneous lung cancer xenografts established by injecting NCI H460 cells (1 x 106/mouse). After 7 days, intratumoral injection of ad-lacZ (1 x 107 pfu), 24RGD (1 x 107 pfu), ad-IGF-1R/482 (1 x 107 pfu), 24RGD (1 x 107 pfu) + ad-lacZ (1 x 107 pfu), and 24RGD (1 x 107 pfu) + ad-IGF1R/482 (1 x 107 pfu) were performed. We repeated the same injections 2 days after the initial injection. Then we measured the change in tumor size by the following formula (0.5 x length x width2).
The tumor growth of each group was analyzed by the repeated ANOVA test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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24RGD.24RGD. Coadministration with 24RGD increased the transduction rate of lacZ from <15% to almost 100% (Fig. 1A)
. This is consistent with the replication of the E1-deleted ad-lacZ with the mutant E1 produced by 24RGD in cancer cells cotransduced with the combination. When we combined ad-luc with 24RGD, the luciferase assay showed an almost 90-fold-increased luciferase expression in 24RGD- and ad-luc-cotransduced cells than in cells transduced with ad-luc alone (Fig. 1B)
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24RGD-Cotransduced Cells.24RGD-cotransduced cells by transferring media conditioned by transduced cells to untransduced cancer cells. Control-conditioned media from NCI H460 cells transduced by ad-lacZ alone transferred lacZ into <1% of cells. However, medium from ad-lacZ- and 24RGD-cotransduced cells transferred lacZ to 20% of cells (Fig. 2)
. This finding confirmed the presence of intact ad-lacZ virions in the media, demonstrating the propagation and release of ad-lacZ from the cotransduced cells. This media-transferable bystander effect of replication-defective adenovirus is a very important characteristic of this strategy.
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24RGD.24RGD, ad-IGF-1R/950 transduction increased the expression of IGF-1R on NCI H460 cells by fluorescence-activated cell sorter from 55.66 to 164.29 (mean values, Fig. 3A
). Western blotting revealed that cotransduction with ad-IGF-1R/482 and 24RGD increased the production of defective subunit in these cells and media by 15.2 and 1.6-fold, respectively, when compared with ad-IGF-1R/482 alone (Fig. 3B)
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24RGD.24RGD (1 x 107 pfu) into palpable tumors, we stained the cut surface of the tumor with X-gal. Figure 4A
shows a representative section. Compared with very low transduction rate by ad-lacZ alone, the combination of ad-lacZ and 24RGD increased the transduction rate (represented by the blue staining area) dramatically.
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24RGD and ad-luc was 70 times higher than in the tumor injected with ad-luc alone. Pertinent to the safety of this approach is that no differences in luciferase expression were observed in the uninjected tumor or vital organs such as liver, spleen, lung, and kidney when compared with ad-luc-injected or -uninjected mice (Fig. 4B)
.
Enhanced In vitro Growth Suppression by Combining 24RGD and ad-IGF-1R/dn.
A single transduction with ad-IGF-1R/482 or 950 showed only minimal growth suppression. 24RGD at a very low dose (0.1 moi) showed a modest and delayed oncolytic effect. However, combined transduction with 24RGD and ad-IGF-1R/482 or 950 induced rapid and strong oncolysis that was evident from day 2 to day 6. By day 4, cells showed almost complete oncolysis. However, 24RGD alone induced complete oncolysis only after day 8 (Fig. 5)
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24RGD.24RGD injection also showed minimal growth suppression at this dose. However, combined injection with 24RGD and ad-lacZ showed some growth suppression. This finding suggests the propagation of ad-lacZ induced enhanced oncolysis although ad-luc itself is not very toxic to cancer cells. Furthermore, 24RGD and ad-IGF-1R/482 combination showed significantly greater growth suppression than the 24RGD and ad-luc combination (P < 0.05, Fig. 6
). Propagation of ad-IGF-1R/482 and subsequent IGF-1 signaling blockade by ad-IGF-1R/482 will be the explanation of this difference.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Armed therapeutic adenoviruses can increase antitumor efficacy by expressing a therapeutic gene, usually in the E1 or E3 region (16 , 21 , 28 , 29) . However, this effect is still restricted to the tumor cells transduced with the oncolytic adenovirus. Therefore, the additional antitumor effect from the inserted gene is not striking.
Our strategy is different from the armed therapeutic adenovirus because we use another replication-defective adenovirus carrying a therapeutic gene instead of inserting the therapeutic gene into a CRAD. Our strategy has a definite advantage over current CRAD and armed therapeutic adenoviruses. This approach induces the propagation of replication-defective adenoviruses carrying a therapeutic gene within tumor cells by using the E1 protein from the cotransduced CRAD. High titers of therapeutic adenovirus, although it is replication-defective itself, can transduce adjacent tumor or stromal cells and kill them even if the CRAD did not infect or would not replicate in those cells. This results in an increase of the transduction efficiency and of the therapeutic efficacy. In other words, we used CRAD-transduced cells as a kind of in situ 293 cell, permitting the propagation of cotransduced E1-deleted and replication-defective adenovirus.
A concern about this approach is the possible competition of CRAD and conventional adenoviruses for infection of the same tumor cell. However, the 24RGD that we used as a CRAD has a RGD motif in the E3 protein that enables this virus to enter the cells without the help of Coxsackie adenoviral receptor. We confirmed this effect by demonstrating an increase in the transduction of lung cancer by ad-lacZ in in vitro (Fig. 1A)
and a 90-fold increase in luciferase expression (Fig. 1B)
by coadministration with 24RGD. This is indirect evidence of ad-lacZ and ad-luc replication by CRAD.
The presence of intact ad-lacZ in the media from the cells transduced with 24RGD and ad-lacZ is direct evidence of ad-lacZ replication and production. This is likely to occur by the replication-defective mutant virus using the E1 (24) protein from 24RGD (Fig. 2)
. This feature is the theoretical basis of the bystander effect observed.
We confirmed this phenomenon again with the therapeutic viruses, ad-IGF-1R/950 and ad-IGF-1R/482. By combining these viruses with 24RGD, ad-IGF-1R/950 induced an increase in IGF-1R expression in tumor cells and ad-IGF-1R/482 induced increased expression of defective subunit in both the transduced cells and, perhaps more importantly, in the media as well.
These findings effectively demonstrate that 24RGD induced the replication of the E1-deleted adenoviruses and induced increased transduction rates and transgene expression.
Safety could be an issue of this strategy because it relies on the propagation of adenovirus in a tumor in situ, and thus there is the possibility of uncontrolled propagation.
Wildner et al. (30) studied the dissemination of replication-competent ad-herpes simplex thymidine kinase after subcutaneous administration. They only found dermatitis and the presence of adenovirus at the injection site without any substantial presence of adenovirus in other organs.
Although a replication-defective therapeutic adenovirus becomes replication competent in this strategy, replication should be restricted to the tumor mass where the CRAD can replicate. The in vivo distribution assay described above using CRAD and ad-luc performed 7 days after injection demonstrated two important findings. The first is that we observed enhanced expression of luciferase from the tumor tissue coinjected with 24RGD and ad-luc. The other important finding is the absence of luciferase expression in the contralateral untreated tumor and other vital organs (lung, liver, spleen, and kidney), even in mice treated with 24RGD and ad-luc. These data suggest that there is no substantial systemic spread of the replication-defective ad-luc. Furthermore, the use of tumor-specific therapeutic genes or tumor-specific promoters may further enhance tumor specificity and avoid uncontrolled proliferation of adenovirus.
The enhanced and tumor-restricted expression of therapeutic genes demonstrated here resulted in an increased treatment efficacy in an animal tumor model in addition to the in vitro growth assay. In this treatment experiment, we used very low dose of ad-IGF-1R/482 (2 x 107 in total). Because of this low dose, ad-IGF-1R/482 alone showed little antitumor effect. 24RGD (2 x 107 in total) showed modest growth suppression. Combination of 24RGD and ad-lacZ also demonstrated a modest antitumor effect by enhanced oncolysis by ad-lacZ propagation. However, the combination of 24RGD and ad-IGF-1R/482 showed very strong tumor suppression over the combination of 24RGD and ad-lacZ. This finding reflected the antitumor effect of ad-IGF-1R/482 that was propagated in tumor mass.
Because these experiments were performed in immunocompromised hosts, anti-adenoviral immune responses in immunocompetent hosts may present another obstacle for viral propagation within the tumor mass if this strategy were applied to the clinic. However, the combination of CRAD and E1-deleted adenovirus with an immune cytokine in the immunocompetent host will be a very attractive combination strategy.
The combination of a CRAD and an E1-deleted adenovirus expressing a therapeutic gene showed a significantly greater antitumor effect although we didn’t compare it directly with an armed CRAD with the same therapeutic gene. Additional experiments will be needed to directly compare the effectiveness of a CRAD + E1-deleted adenovirus with a therapeutic gene and an armed therapeutic CRAD.
Thus we have identified a novel gene-therapeutic approach that significantly improved gene transfer and gene therapeutic efficacy in model tumors in vivo and that has potential for direct clinical applicability for a variety of tumor types and therapeutic genes.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: David P. Carbone, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232-6838. Phone: 615-936-3321; Fax: 615-936-3322; E-mail: d.carbone{at}vanderbilt.edu
Received 4/ 5/04. Revised 6/20/04. Accepted 7/22/04.
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, Control; 
, Ad-luc; 
, CRAD+Ad-luc.
, Control;
, IGF-1R/482;
, IGF-1R/950;
, CRAD;
, CRAD+IGF-1R/482;
, CRAD+IGF-1R/950.
, CRAD+Ad-IGF-1R/482.