Interleukin 12 and B7-1 costimulatory molecule ... - Semantic Scholar

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We thank U. Sankar and J. Rudy for technical assistance, H. Liang for flow cytometry ... S. C., Gubler, U., Wolf, S. F., Robbins, P. D. & Lotze, M. T.. (1994) Cancer ...
Proc. Natl. Acad. Sci. USA Vol. 94, pp. 10889–10894, September 1997 Medical Sciences

Interleukin 12 and B7-1 costimulatory molecule expressed by an adenovirus vector act synergistically to facilitate tumor regression (gene therapyyimmunotherapyycytokinesycancer)

¨ TZER*, MARY HITT*, WILLIAM J. MULLER†, PETER EMTAGE†, JACK GAULDIE†, BRIGITTE M. PU AND FRANK L. GRAHAM*†‡ Departments of *Biology and †Pathology, McMaster University, 1280 Main Street West, Hamilton, ON Canada L8S 4K1

Communicated by C. Thomas Caskey, Merck & Co., West Point, PA, August 6, 1997 (received for review April 7, 1997)

mediate tumor rejection, and in some cases systemic immunity (8), whereas several other poorly immunogenic tumors failed to induce an appropriate immune response (9). B7-1 mediated antitumor activity was largely attributed to its ability to stimulate natural killer (NK) and CD81 T cells, whereas the requirement of CD41 cells for tumor rejection is highly dependent on the tumor model (8, 10, 11). In addition to membrane-bound costimulators, soluble cytokines produced by antigen-presenting cells play a key role in regulating T cell activity. Among these, IL-12 has attracted particular attention for its antitumor properties (12–15). IL-12 is a heterodimeric protein (16) produced by activated monocyte-macrophages and B lymphocytes which augments proliferation of activated NK and cytotoxic T cells, stimulates interferon g production by NK and T cells, and enhances lymphokine-activated killer activity (17). Although expression of either IL-12 or B7-1 alone has been shown to be effective in tumor regression and induction of protective immunity against certain tumors, their therapeutic benefit is limited. Current evidence suggests that the generation of a tumor milieu containing both increased levels of certain cytokines and the presence of costimulatory molecules might be a promising approach to potentiate the antitumor efficacy over that attainable with either one alone. In this paper, we describe construction of a recombinant adenovirus 5 (Ad5) carrying both murine (m) IL-12 subunits in early region 1 (E1) and the mB7-1 cDNA in E3 (AdIL12–B7-1), each under control of the murine cytomegalovirus (MCMV) promoter. We show that this vector expressed high levels of functional IL-12 and B7-1 in infected cells and was extremely efficient in inducing regression of established tumors in a transgenic mouse model of metastatic breast cancer, resulting in a protective immunity against a second challenge with tumor cells. These results indicate that Ad-based vectors should afford a very effective system to deliver synergistically interacting proteins for in vivo gene therapy.

ABSTRACT Stimulation of antitumor immune mechanisms is the primary goal of cancer immunotherapy, and accumulating evidence suggests that effective alteration of the host–tumor relationship involves immunomodulating cytokines and also the presence of costimulatory molecules. To examine the antitumor effect of direct in vivo gene transfer of murine interleukin 12 (IL-12) and B7-1 into tumors, we developed an adenovirus (Ad) vector, AdIL12–B7-1, that encodes the two IL-12 subunits in early region 1 (E1) and the B7-1 gene in E3 under control of the murine cytomegalovirus promoter. This vector expressed high levels of IL-12 and B7-1 in infected murine and human cell lines and in primary murine tumor cells. In mice bearing tumors derived from a transgenic mouse mammary adenocarcinoma, a single intratumoral injection with a low dose (2.5 3 107 pfuymouse) of AdIL12–B7-1 mediated complete regression in 70% of treated animals. By contrast, administration of a similar dose of recombinant virus encoding IL-12 or B7-1 alone resulted in only a delay in tumor growth. Interestingly, coinjection of two different viruses expressing either IL-12 or B7-1 induced complete tumor regression in only 30% of animals treated at this dose. Significantly, cured animals remained tumor free after rechallenge with fresh tumor cells, suggesting that protective immunity had been induced by treatment with AdIL12–B7-1. These results support the use of Ad vectors as a highly efficient delivery system for synergistically acting molecules and show that the combination of IL-12 and B7-1 within a single Ad vector might be a promising approach for in vivo cancer therapy. Progressive tumor growth in immunocompetent hosts suggests that immune mechanisms are insufficient to detect and eliminate malignant cells. Recent studies indicate that this failure is not always due to the lack of expression of tumor antigens, but rather results from the inability of tumor antigens bound to the major histocompatibility complex (Ag-MHC) to induce adequate T cell proliferation or effector function (1). In this context, it has been shown that the activation of a helper T cell to produce sufficient interleukin 2 (IL-2) to allow autocrinedriven clonal expansion requires costimulatory or accessory signals in addition to T cell receptor (TCR) ligation by Ag-MHC (2–4). The interaction of the CD28 receptor constitutively expressed on T cells with B7-1, a membrane glycoprotein that is induced upon activation on various antigenpresenting cells (5–7), represents one of the best-characterized examples of such a costimulatory signal. Introduction of the B7-1 gene into a variety of murine tumors has been shown to

MATERIALS AND METHODS Construction of Recombinant Plasmids and Adenoviral Vectors. Plasmids were constructed by standard protocols (18). Plasmid DNA was prepared by the alkaline lysis method (19) and purified by CsCl-ethidium bromide density gradient centrifugation. The mIL-12 p35 and p40 cDNAs were obtained from the plasmids pVL2IL12p35 and pMEIL12p40#4, respectively (kindly provided by A. O’Garra, DNAX). The XbaIy Abbreviations: Ag-MHC, antigen bound to the major histocompatibility complex; TCR, T cell receptor; IL, interleukin; Ad, adenovirus; E, early region; MCMV, murine cytomegalovirus; HCMV, human cytomegalovirus; m, murine; FACS, fluorescence-activated cell sorter; p.i., postinfection. ‡To whom reprint requests should be addressed. e-mail: graham@ mcmaster.ca.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y9410889-6$2.00y0 PNAS is available online at http:yywww.pnas.org.

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BglII cDNA fragment encoding murine B7-1 from 250 to 11,082 was generated by reverse transcription–PCR amplification. The expression cassette for IL-12 was inserted in the E1 region of pMH4 (20). IL-12 subunits, p40 and p35, were separated by the encephalomyocarditis virus internal ribosome entry site and expressed from a polycistronic message under control of the short MCMV immediate early promoter and terminated by the polyadenylation signal of simian virus 40 (pMEM12R; unpublished data). Ads expressing foreign genes were obtained by homologous recombination using the pBHG10 system in 293 cells (21, 22). The virus AdMEM12R contains the expression cassette for both IL-12 subunits in E1 and a deletion (78.3–85.8 mu) in E3 (M.H. and F.L.G., unpublished results). The control Ad, dl70-3, is a mutant of Ad5 deleted in E1 and having a deletionysubstitution in E3 (21). Cell Culture. The 293 cell line (Ad E1-transformed human embryonic kidney cells; ref. 23) was maintained in F-11 medium supplemented with 10% fetal bovine serum (FBS). MRC5 cells (human fibroblasts; ATCC CCL 171), A549 cells (human lung carcinoma; ATCC CCL 185), and WN35 (B72 human melanoma cell line; generated by M. Herlyn, Pittsburgh, and provided by S. Dessureault) were grown in aminimal essential medium plus 10% FBS. MT1A2 cells (20) were derived from a mammary adenocarcinoma isolated from a transgenic mouse carrying the polyoma middle T (PyMidT) gene under the control of the murine mammary tumor virus LTR (24). MT1A2 and primary FVBMT cells, prepared by explanting tumor tissue from tumor-bearing PyMidT transgenic mice, were grown in F-11 medium with 10% FBS. All culture media were supplemented with 2 mM L-glutamine, 100 mgyml penicillin, and 100 unitsyml streptomycin. IL-12 ELISA and Proliferation Assay. Cells were infected with the appropriate virus at a multiplicity of infection (moi) of 10 plaque forming units (pfu) per cell and at various times postinfection (p.i.) aliquots of infected cell supernatant were removed and stored at 220°C for assays of mIL-12. Expression levels of secreted mIL-12 were quantitated by ELISA as described (25) using a polyclonal rabbit antiserum that recognizes both subunits of mIL-12 (gift of Steven Kunkel, University of Michigan, Ann Arbor). Biological activity was tested by assaying the proliferation of Con A-activated FVByn mouse splenocytes in response to IL-12 (26). The mIL-12 concentration for each sample was determined by comparison to a standard curve prepared using recombinant mIL-12 (gift of A. O’Garra, DNAX). One unit of mIL-12 is defined as the amount of IL-12 producing half-maximal proliferation of the Th1 clone HDK1 (27). Five units of IL-12 are equivalent to 1 ng IL-12 protein. Immunostaining and Flow Cytometry Analysis (Fluorescence-Activated Cell Sorter; FACS). For B7-1 detection, cells infected at a moi of 10 were incubated in 5 ml growth medium for various times, trypsinized, and resuspended in ice cold PBS with 1% FBS. Cells were incubated with the CD80 Biotin anti-mouse B7-1 antibody (PharMingen) for 30 min at 4°C, washed with PBS containing 1% BSA, and incubated for an additional 15 min at 4°C with streptavidin, R-phycoerythrin conjugate (Molecular Probes). After another wash the cells were resuspended in PBS containing 1% BSA. Cells were analyzed on a FACScan flow cytometer (Becton Dickinson). Intratumoral Injection with Ad Vectors and Rechallenge of Tumor-Free Animals. The transgenic PyMidT model and tumor establishment in syngenic FVByn mice have been described (28). After developing visible tumors ('21 days p.i. with 106 tumor cell) mice were injected intratumorally with the appropriate concentration of virus in PBS. Tumors were measured prior to virus injection and subsequently at twice weekly intervals using calipers, and the tumor volume was calculated from the longest diameter and average width, assuming a prolate spheroid (28). Animals were killed when

Proc. Natl. Acad. Sci. USA 94 (1997) the longest diameter was greater than 15 mm or when any two measurements were greater than 10 mm. Two to 3 months after complete regression of primary tumors mice were rechallenged with freshly isolated tumor cells by s.c. injection of 106 cells in the opposite flank.

RESULTS Construction of AdIL12–B7-1. The construction of AdIL12– B7-1 was based on the Ad5 recombinant system that allows insertion of expression cassettes in either E1 or E3 (21). A previously constructed Ad vector, AdmIL12.1, contains the p35 subunit cDNA of IL-12 in E1 and the cDNA for p40 in E3 under control of the human CMV (HCMV) promoter and the polyadenylation signal of simian virus 40 (29). To derive a vector expressing both IL-12 and B7-1, we inserted both mIL-12 subunit cDNAs p40 and p35 in the E1 region and placed them under control of the MCMV promoter, recently shown to drive much higher levels of transgene expression than the HCMV promoter (20). To achieve efficient expression of both IL-12 subunits, we used the internal ribosome entry site sequence from encephalomyocarditis virus that allows initiation of translation at downstream start codons (17, 30). To introduce the coding sequence for B7-1 (250 to 11,082) in the E3 region of pBHG10 (21), an XbaIyBglII B7-1 cDNA fragment was first subcloned into the NheI and BamHI site of pMH4 (20), generating pMCMVB7, which contains the B7-1 cDNA flanked by the MCMV promoter (from 2454 to 132 relative to the transcription start site for the MCMV IE gene) and the simian virus 40 poly(A). The expression cassette was cleaved from pMCMVB7 by XbaIyBglII and inserted into XbaIyBamHI restricted pABS.4 (contains the kanamycin resistance gene, kanr) yielding pABS.B7-1. The B7-1 cassette plus kanamycin was excised from pABS.B7-1 by partial PacI digestion and ligated into PacI restricted pBHG10. This final step generated pBHG10-B7-1 carrying the B7 expression cassette with kanr in the E3 region of pBHG10 in parallel orientation with E3 transcription. AdIL12–B7-1 was generated by cotransfection of 293 cells with pBHG10 –B7-1 and pMEM12R (Fig. 1). In addition, pBHG10–B7-1 was cotransfected with pDE1Sp1A (E1 deletion 0.9–9.8 mu; ref. 21) generating AdDE1–B7-1 that has a deletion in E1 and the B7-1 cassette in E3 (Fig. 1). Characterization of AdIL12–B7-1. To assay IL-12 expression, three different cell lines were infected with AdIL12–B7-1 and expression levels were quantitated by ELISA (Fig. 2A). Murine and human carcinoma cells as well as human fibroblasts were found to secrete high amounts of IL-12. After 5

FIG. 1. Structure of AdIL12–B7-1. The expression cassette for IL-12 replaces E1 of the Ad5 genome. The IL-12 subunit cDNAs, IL-12p40 and IL-12p35, are separated by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) and are expressed from a polycistronic message. The B7-1 cDNA is inserted in E3. Both foreign genes are under control of the MCMV IE promoter (2454 to 132 relative to the transcription start site) and terminated by the polyadenylation signal of simian virus 40. The Ad vector was obtained by homologous recombination using the pBHG10 vector system (21). Two additional vectors containing either the IL-12 expression cassette in E1 (AdMEM12R) or the B7 cassette in E3 (AdDE1-B7-1) were also constructed.

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FIG. 2. Expression kinetics and biological activity of IL-12 in AdIL12–B7-1-infected cells. The levels of IL-12 secretion following infection with AdIL12–B7-1 (moi 10) were determined by ELISA. (A) IL-12 expression in MRC5 (h), A549 ({), and MT1A2 (‚). (B) Comparison of IL-12 levels in AdIL12–B7-1 (h), AdMEM12R ({), and AdmIL12.1 ( ‚ ) infected primar y FVBMT tumor cells. AdDE1B7-1 (E) was used as a negative control. (C) Biological activity of IL-12 was tested by assaying the proliferation of Con A-activated mouse splenocytes in response to IL-12. FVByn-mouse splenocytes (106 cellsyml) were incubated with supernatants from AdIL12–B7-1 and AdDE1B7-1 infected MT1A2 (72 h p.i.) and primary FVBMT cells (48 h p.i.) for 24 h. After overnight incubation with [3H]thymidine, incorporation was measured by liquid scintillation counting. The IL-12 sample concentration was determined by comparison with a standard curve prepared using recombinant mIL-12. Five units of IL-12 are equivalent to 1 ng IL-12 protein. All samples were assayed in triplicate.

days, expression in human A549 cells reached 9 mg IL-12 and 3 mg IL-12 in MRC5 per 106 infected cells. Much higher levels of expression were detected in infected murine breast cancer cells derived from transgenic tumors. As shown in Fig. 2 A and B, the highest amount of IL-12 was detected in AdIL12–B71-infected MT1A2 (60 mgy106 infected cells at day 5) and FVBMT cells (60 mgy106 cells at day 3). Compared with the Ad vector described in ref. 29, we attained a 6-fold higher level of

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IL-12 expression by the MCMV promoter in FVBMT cells (Fig. 2B). Notably, in primary tumor cells IL-12 levels induced by AdIL12–B7-1 infection were comparable to those produced by AdMEM12R, indicating that the presence of a second gene in E3 did not affect protein expression from a transgene inserted in E1. AdDE1B7-1, used as a control, showed no IL-12 expression. Supernatants from infected MT1A2 (72 h p.i.) and primary FVBMT cells (48 h p.i.) were used at 3-fold serial dilutions to verify IL-12 bioactivity by its ability to stimulate proliferation of Con A-activated splenocytes. A strong stimulation of the target cells was observed using supernatants from both AdIL12–B7-1-infected cell lines, whereas the supernatants from AdDE1B7-1-infected cultures had no stimulatory effect (Fig. 2C). The level of bioactive IL-12 product in FVBMT cells infected with AdMEM12R was similar to that obtained with AdIL12–B7-1 infections (data not shown). Thus, IL-12 bioactivity correlated with the level of secreted IL-12 protein in both AdIL12–B7-1- or AdMEM12R-infected cell lines. Furthermore, immunoprecipitation analysis indicated that equal amounts of p35 and p40 were made in infected cells (data not shown). Together these data suggest a balanced expression of both IL-12 subunits in AdIL12–B7-1-infected cells, previously demonstrated to be important for its biological activity (31). To confirm that mB7-1 was being expressed, human WN35 and mouse MT1A2 cells were infected with AdIL12–B7-1 or AdDE1B7-1 and infected cells were harvested 24 and 72 h later and assayed for B7-1 by FACS. As shown in Fig. 3A, both cell lines infected with AdIL12–B7-1 reacted strongly with B7-1 antibody [after 72 h 78% (WN35) and 93% (MT1A2) were positive with a mean fluorescence of 432 and 576, respectively]. B7-1 was also strongly presented on the surface of WN35 and MT1A2 cells infected by AdDE1B7-1 [after 72 h 77% (WN35) and 97% (MT1A2) were positive with a mean fluorescence of 425 and 526, respectively], indicating that the B7-1 gene was expressed by each of these vectors and the product was translocated correctly (Fig. 3B). Uninfected WN35 cells do not react with B7-1 antibody. In addition, FACS analysis showed that uninfected MT1A2 cells, which scored almost negative for B7-1 expression (4% after 72 h), were positive for the expression of MHC class I (data not shown). Because previous data have indicated that an adequate immune response depends not only on TCR ligation by Ag-MHC, but also requires an efficient costimulatory signal (2–4), our mammary adenocarcinoma model represents a highly appropriate model to examine the effect of B7-1 overexpression on the surface of tumor antigen presenting cells for induction of an efficient antitumor T cell response. Regression of Established Tumors Following Single Intratumoral Injections. We have previously demonstrated that intratumoral injection with 5 3 108 pfu of an Ad vector expressing IL-2 or IL-12 can induce tumor regression in 30–50% of treated animals (28, 32). To determine the relative antitumor activity of Ad vectors expressing either IL-12 or B7-1 alone compared with the virus encoding both genes, we initially injected tumors with low doses (2.5 3 107 pfu) of either AdDE1B7-1, AdMEM12R, AdIL12–B7-1, or the Addl70-3 control virus and monitored the mice for tumor regression. Representative data from one experiment (experiment 1 in Table 1) are shown for tumor volume (Fig. 4A) and for survival (Fig. 4B) as a function of time after treatment. At 2.5 3 107 pfu AdIL12–B7-1 treatment caused a significant regression from a mean tumor volume of 58 6 13 mm3 at the time of injection to 11 6 7 mm3 by day 14 (Fig. 4A). At this virus dose, reduction in tumor volume was not detectable following injection of the B7-1 or IL-12 virus alone, but these treatments resulted in a pronounced growth delay and prolonged survival compared with Addl70-3-injected animals (Fig. 4B). Following intratumoral injection with Addl70-3 all tumors continued to grow until the animals had to be sacrificed at day 24 (Fig. 4).

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Proc. Natl. Acad. Sci. USA 94 (1997) Table 1. Ability of AddlE1–B7-1, AdMEM12R and AdIL12–B7-1 to induce tumor regression following a single intratumoral injection Response Virus AddlE1–B7-1 (2.5 3 107)

AdMEM12R (2.5 3 107)

AddlE1–B7-1 AdMEM12R (2.5 3 107) AdIL12–B7-1 (2.5 3 107)

AdIL12–B7-1 (1 3 107) AdIL12–B7-1 (1 3 108)

Exp. None* Growth delay† Partial‡ Complete§ 1 0 2 1 3 0 Total (1y15) 1 1 2 1 3 0 Total (2y15) 1 0 2 0 Total 1 0 2 1 3 0 Total (1y20) 1 0 2 0 Total 1 0 2 0 Total

5 4 5 (14y15) 4 4 5 (13y15) 2 4 (6y10) 1 1 3 (5y20) 3 2 (5y10) 0 0

0 0 0

0 0 0

0 0 0

0 0 0

1 0 (1y10) 0 0 0

2 1 (3y10) 4 3 7 (14y20) 0 0

2 3 (5y10) 0 1 (1y10)

5 4 (9y10)

*Tumor development following injection not significantly different from mice treated with control Addl70-3. †Slight decrease in tumor volume andyor tumor growth significantly delayed and animals survived 2-8 weeks longer than controls. ‡Tumors completely regressed for at least 2 weeks. §Tumors completely regressed and did not reoccur.

FIG. 3. Expression of B7-1 on tumor cells analyzed by FACS. Levels of B7-1 expressed by (A) AdIL12–B7-1 or (B) AdDE1B7-1 (moi 10) on the surface of MT1A2 and WN35 cells (black peaks) relative to the uninfected control (white peaks) were determined 24 and 72 h after infection. Cells were stained with CD80 Biotin anti-mouse B7-1 antibody and streptavidin, R-phycoerythrin conjugate, and subjected to FACS analysis as described.

AdDE1B7-1-treated mice survived 2–3 weeks longer, and AdMEM12R-treated mice 6 weeks longer than the control group, but in all three groups 100% of the tumors grew progressively. In contrast, treatment with AdIL12–B7-1 produced complete tumor regression in four of five mice, and animals that underwent complete regression remained tumor free (Fig. 4B). Even after injection of a lower dose of AdIL12– B7-1 (1 3 107 pfu), 5y10 tumors showed a partial response and totally disappeared for at least 2 weeks before reoccurrence. In the remaining 5y10 animals tumor development was significantly delayed and survival was increased, similar to AdMEM12R-injected mice, for up to 6 weeks longer than controls (Fig. 4). The results presented in Fig. 4 and the data from two additional experiments are summarized in Table 1. The combined data from three experiments show that although the majority of animals treated in these experiments with either AdMEM12R or AdDE1B7-1 responded to virus injection by a delay in tumor growth (87% and 93%, respectively) and prolongation of survival, none of the mice showed evidence of complete tumor regression. When tumors were coinjected with AdMEM12R and AdDE1B7-1, each at a dose of 2.5 3 107 pfu, complete regression was induced in 3y10

tumors and 1y10 animals partially regressed over 2 weeks (Table 1). In contrast, treatment with 2.5 3 107 pfu AdIL12– B7-1 induced complete tumor regression in 14y20 tumors (70%) and in the remaining 6 animals 5 tumors showed a pronounced delay in tumor growth lasting 2–8 weeks. Overall, only 1y30 animals injected with AdIL12–B7-1 at two different doses failed to show a response. At a higher dose of AdIL12– B7-1 (1 3 108 pfu) 9 of 10 treated mice showed complete regression and the one remaining animal had a partial regression lasting 3 weeks (Table 1). By contrast, injection of Ad vectors expressing B7-1 or IL-12 alone at 1 3 108 pfu resulted only in partial regression. Together our data argue that coexpression of B7-1 and IL-12 from a single vector can act synergistically to facilitate tumor regression. It should be mentioned that high doses of AdMEM12R (5 3 108) resulted in toxicity and death within 6–10 days p.i. In contrast, no obvious toxic effects were observed in mice treated with AdIL12–B7-1 at the doses that resulted in cures in most mice. AdIL12–B7-1 Induced Tumor Regression Caused LongTerm Antitumor Immunity. To test whether mice successfully treated with AdIL12–B7-1 were protected against a second inoculation with tumor cells, we rechallenged 10 tumor-free animals 2–3 months following complete primary tumor regression by injecting 106 fresh tumor cells s.c. into the opposite flank. No tumors were induced in any of the mice, whereas 100% of age-matched syngeneic control animals developed tumors within 21 day after injection. These results indicate that AdIL12–B7-1 administration is associated with long-term antitumor immunity which might inhibit tumor reocurrence and prevent metastasis.

DISCUSSION IL-12 is a heterodimeric protein consisting of two subunits of 35 and 40 kDa (16, 30), and coexpression of both subunit cDNAs in cultured cells has been shown to be necessary for the secretion of biologically active protein (30). IL-12 is normally

Medical Sciences: Pu ¨tzer et al.

FIG. 4. Regression of established tumors following a single intratumoral injection of AdIL12–B7-1. Tumors were injected with 2.5 3 107 pfu of AdDE1B7-1, AdMEM12R, and two different doses of AdIL12–B7-1 (2.5 3 107 and 1 3 107 pfu) or the control virus Addl70-3. (A) Mean tumor volume of mice measured every 4–5 days: h, Addl70-3 (n 5 5); E, AdDE1B7-1 (n 5 5); ‚, AdMEM12R (n 5 5); m, AdIL12–B7-1 (1 3 107; n 5 5); Œ, AdIL12–B7-1 (2.5 3 107; n 5 5). (B) Survival of mice following treatment with the viruses shown in A. Symbols are as shown in A.

produced by activated monocyte-macrophages and B lymphocytes, and receptors for IL-12 are present on activated CD561 NK cells and activated CD41 and CD81 T cells (33). It stimulates activated NK and T cells to produce high levels of interferon g, increases NKylymphokine-activated killer activity as shown in studies with a retrovirus expressing human IL-12 (17), and appears to be a major determinant in the development of Th1yTC1 response (34). B7-1 is expressed on activated B cells and other professional antigen-presenting cells. The interaction of B7-1 with its natural ligand CD28 on T cells in combination with TCR occupancy results in T cell stimulation and lymphokine secretion, as shown by the ability of B7-1-transfected cell lines to serve as a cofactor for IL-2 production by stabilizing IL-2 mRNA (35), and leads to increased cytolytic activity of CD81 cells (36) and CD41 T cell proliferation in vitro (37). In addition, B7yCD28 interaction has been demonstrated to prevent long-term T cell unresponsiveness or anergy resulting from TCR ligation in the absence of costimulatory signal (38–40).

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In vivo, genetic modification of a variety of murine tumors for local expression of either B7-1 or IL-12 has previously been demonstrated to render tumor cells susceptible to T cellmediated rejection resulting in tumor regression and in some cases establishment of systemic immunity (8, 41–43). We previously generated an Ad vector with insertions of the mIL-12 p35 and p40 subunit cDNAs in E1 and E3, respectively, driven by the HCMV promoter (29). A single injection with 5 3 108 pfu of this vector induced complete tumor regression in 30% of established tumors in a murine breast cancer model (32). In the present study we examined the ability of the single Ad vector expressing both IL-12 and B7-1 to promote tumor regression following injection at 20-fold lower virus doses (2.5 3 107 pfu) and compared the antitumor effect with a vector encoding either IL-12 or B7-1 alone, or the results of coinjection of the two Ad vectors. Whereas neither B7-1 nor IL-12 expression in tumor cells by itself induced regression, 30% of the tumors completely regressed after coinjection of both Ad vectors, indicating that IL-12 and B7-1 act synergistically to promote an effective antitumor response. The observed immune response is most likely based on a cooperative interaction between both immunomodulatory molecules in inducing efficient proliferation and interferon g production of T cells which has been described in vitro (27, 44) and in vivo by lymphocyte ablation studies (45). Although coinjection of tumors with two vectors each expressing IL-12 or B7-1 was more effective than injection with either vector alone, treatment with the single vector coexpressing both IL-12 and B7-1 was consistently the most effective. Maximal synergy may require a high concentration of IL-12 in the environment of B7-1-positive tumor cells. This synergy is more likely to occur following injection of a single vector containing both IL-12 and B7-1 expression units than with two separate viruses, that would probably not coinfect the same cells with high efficiency following in vivo administration. A decrease of the AdIL12– B7-1 viral load to 1 3 107 pfu still induced a strong antitumor response but did not result in complete regression, whereas 90% of the tumors completely regressed after injection of 1 3 108 pfu, a dose that does not lead to toxicity. In contrast, AdMEM12R, the vector expressing IL-12 alone, exhibited toxic effects at doses needed to induce regression (unpublished data). Interestingly, an earlier vector expressing IL12, AdmIL12.1 (29, 32), was completely nontoxic at all concentrations tested (up to 1 3 109 pfu) and therefore differed significantly from AdMEM12R. There are at least three possible explanations for the toxic effects observed with AdMEM12R: the higher IL-12 expression level induced by the MCMV promoter in this vector compared with our earlier construct (29, 32), differences in the kinetics of expression [vectors containing transgenes under the control of the MCMV promoter exhibit an earlier peak of expression than those with the HCMV promoter (20)], and finally differences in relative expression of the p35 and p40 subunits [equal amounts from AdMEM12R versus an excess of p40 protein from AdmIL12.1 (29)]. Although recent reports have shown that IL-12 and B7-1 cooperate in inducing an antitumor effect (27, 44–47), these studies have employed ex vivo transduction of tumor or fibroblast cells by retrovirus vectors and injection of the engineered cells or have used systemic administration of a potentially toxic recombinant cytokine. We believe that use of a single intratumoral injection of an Ad vector expressing both genes has advantages of greater simplicity and efficacy over other approaches. Our findings provide evidence for protective immunity in mice where tumors completely regressed following injection of AdIL12–B7-1. In rechallenge experiments 100% of the mice were protected against a second tumor cell administration, indicating that these mice developed long-term immunity. A complete immunity was also observed following tumor regression with an Ad vector expressing IL-2 at 5 3 108

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pfu (28), whereas not all mice cured with the same dose of an IL-12 expressing virus possessed long-term immunity (32). However, in both cases complete regression correlated with increased tumor-specific cytotoxic T cell activity (C. Addison, J. L. Bramson, M.H., J.G., and F.L.G., unpublished data). Activated tumor-specific cytotoxic T cells were also found in completely regressed mice in response to AdIL12–B7-1 immediately and 3 months after tumor regression (data not shown). The present study provides further support for the use of Ad vectors in cancer therapy and particularly for delivery of cooperatively interacting immunomodulatory genes in a single vector. Combined high level expression of IL-12 and B7-1 greatly increased the frequency of complete regression of established tumors, compared with vectors expressing either cytokine alone, and resulted in long-lasting antitumor immunity at a low vector concentration that is not associated with systemic toxicity. Taken together, our results suggest that Ad vectors expressing IL-12 and B7-1 in combination might represent a highly effective strategy for in vivo therapy of patients with metastatic cancer.

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17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

We thank U. Sankar and J. Rudy for technical assistance, H. Liang for flow cytometry analysis, R. J. Parks for critical reading of the manuscript, and C. L. Addison and B. Marr for helpful advice. This work was supported by grants from the National Cancer Institute of Canada, the Medical Research Council of Canada, the Canadian Breast Cancer Initiative, Baxter Healthcare, and London Life Insurance. B.M.P. is supported by a fellowship from the Deutsche Krebshilfe, Mildred Scheel Stiftung. F.L.G. is a Terry Fox Research Scientist and W.J.M. is a Medical Research Council scientist of the National Cancer Institute of Canada. All animal work has been approved by and carried out according to guidelines set by the Animal Research Ethics Board of McMaster University. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Boon, T., Cerottini, J., Van den Eynde, B., van der Bruggen, P. & Van Pel, A. (1994) Annu. Rev. Immunol. 12, 337–365. Mueller, D. J., Jenkins, M. K. & Schwartz, R. H. (1989) Annu. Rev. Immunol. 7, 445–480. Liu, Y. & Linsley, P. S. (1992) Curr. Opin. Immunol. 4, 265–270. June, C. H., Bluestone, J. A., Nadler, L. M. & Thompson, C. B. (1994) Immunol. Today 15, 321–331. Freeman, G. J., Gray, G. S., Gimmi, C. D., Lombard, D. B., Zhou, L.-J., White, M., Fingeroth, J. D., Gribben, J. G. & Nadler, L. M. (1991) J. Exp. Med. 174, 625–631. Azuma, M., Yssel, H., Phillips, J. H., Spits, H. & Lanier, L. L. (1993) J. Exp. Med. 177, 845–850. Zitvogel, L., Mayordomo, J. I., Tjandrawan, T., DeLeo, A. B., Clarke, M. R., Lotze, M. T. & Storkus, W. J. (1996) J. Exp. Med. 183, 87–97. Wu, T. C., Huang, A. Y., Jaffee, E. M., Levitsky, H. I. & Pardoll, D. M. (1995) J. Exp. Med. 182, 1415–1421. Chen, L., McGowan, P., Ashe, S., Johnston, J., Li, Y., Hellstro ¨m, I. & Hellstro ¨m, K. E. (1994) J. Exp. Med. 179, 523–532. Geldhof, A. B., Raes, G., Bakkus, M., Devos, S., Thielemans, K. & De Baetselier, P. (1995) Cancer Res. 55, 2730–2733. Li, Y., McGowan, P., Hellstro ¨m, I., Hellstro ¨m, K. E. & Chen, L. (1994) J. Immunol. 153, 421–428. Brunda, M. J., Luistro, L., Warrier, R. R., Wright, R. B., Hubbard, B. R., Murphy, M., Wolf, S. F. & Gately, M. K. (1993) J. Exp. Med. 78, 1223–1230. Tahara, H., Zeh,H. J., III, Storkus, W. J., Pappo, I., Watkins, S. C., Gubler, U., Wolf, S. F., Robbins, P. D. & Lotze, M. T. (1994) Cancer Res. 54, 182–189. Tahara, H., Zitvogel, L., Storkus, W. J., Zeth, III, H. G., McKinney, T. G., Schreiber, R. D., Gubler, U., Robbins, P. D. & Lotze, M. T. (1995) J. Immunol. 154, 6466–6474. Rakhmilevich, A. L., Turner, J., Ford, M. J., McCabe, D., Sun, W. H., Sondel, P. M., Grota, K. & Yang, N.-S. (1996) Proc. Natl. Acad. Sci. USA 93, 6291–6296. Wolf, S. F., Temple, P. A., Kobayashi, M., Young, D., Dicig, M., Lowe, L., Dzialo, R., Fitz, L., Hewick, R. M., Kelleher, K.,

27. 28. 29. 30. 31.

32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.

Herrmann, S. H., Clark, S. C., Azzoni, L., Chan, S. H., Trinchieri, G. & Perussia, B. (1991) J. Immunol. 146, 3074–3081. Zitvogel, L., Tahara, H., Cai, Q., Storkus, W., Muller, G., Wolf, S. F., Gately, M., Robbins, P. D. & Lotze, M. T. (1994) Hum. Gene Ther. 5, 1493–1506. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, NY), 2nd Ed., pp. 1.21–1.84. Birnboim, H. C. & Doly, J. (1979) Nucleic Acids Res. 7, 1513– 1523. Addison, C. L., Hitt, M., Kunsken, D. & Graham, F. L. (1997) J. Gen. Virol., in press. Bett, A. J., Haddara, W., Prevec, L. & Graham, F. L. (1994) Proc. Natl. Acad. Sci. USA 91, 8802–8806. Hitt, M., Bett, A. J., Prevec, L. & Graham, F. L. (1994) in Cell Biology: A Laboratory Handbook, ed.Celis, J. E. (Academic, San Diego), pp. 479–490. Graham, F. L., Smiley, J., Russell, W. C. & Nairn, R. (1977) J. Gen. Virol. 36, 59–72. Guy, C. T., Cardiff, R. D. & Muller, W. J. (1992) Mol. Cell. Biol. 12, 954–961. Chensue, S. W., Ruth, J. H., Warmington, K., Lincoln, P. & Kunkel, S. L. (1995) J. Immunol. 155, 3546–3551. Schoenhaut, D. S., Chua, A. O., Wolitzky, A. G., Quinn, P. M., Dwyer, C. M., McComas, W., Familletti, P. C., Gately, M. K., and Gubler, U. (1992) J. Immunol. 148, 3433–3440. Murphy, E. E., Terres, G., Macatonia, S. E., Hsieh, C. S., Mattson, J., Lanier, L., Wysocka, M., Trinchieri, G., Murphy, K. & O’Garra, A. (1994) J. Exp. Med. 180, 223–231. Addison C. L., Braciak, T., Ralston, R., Muller W. J., Gauldie, J. & Graham F. L. (1995) Proc. Natl. Acad. Sci. USA 92, 8522–8526. Bramson, J., Hitt, M., Gallichan, W. S., Rosenthal, K. L., Gauldie, J. & Graham F. L. (1996) Hum. Gene Ther. 7, 333–342. Ghattas, I. R., Sanes, J. R. & Majors, J. E. (1991) Mol. Cell. Biol. 11, 5848–5859. Gately, M. K., Carvajal, D. M., Connaughton, S. E., Gillessen, S., Warrier, R. R., Kolinsky, K. D., Wilkinson, V. L., Dwyer, C. M., Higgins, G. F., Jr., Podlaski, F. J., Faherty, D. A., Familletti, P. C., Stern, A. S. & Presky, D. H. (1996) Ann. N.Y. Acad. Sci. 795, 1–12. Bramson, J. L., Hitt, M., Addison, C. L., Muller, W. J., Gauldie, J. & Graham, F. L. (1996) Hum. Gene Ther. 7, 1995–2002. Desai, B. B., Quinn, P. M., Wolitzky, A. G., Mongini, P. K., Chizzonite, R. & Gately, M. K. (1992) J. Immunol. 148, 3125– 3132. Hendrzak, J. A. & Brunda, M. J. (1995) Lab. Invest. 72, 619–637. Linsley, P. S., Brady, W., Grosmaire, L., Aruffo, A., Damle, N. K. & Ledbetter, J. A. (1991) J. Exp. Med. 173, 721–730. Harding, F. A. & Allison, J. P. (1993) J. Exp. Med 177, 1791–1796. Gajewski, T. F. (1996) J. Immunol. 156, 465–472. Harding, F. A., McArthur, J. G., Gross, J. A., Raulet, D. H. & Allison, J. P. (1992) Nature (London) 356, 607–609. Gimmi, C. D., Freeman, G. J., Gribben, J. G., Gray, G. & Nadler, L. M. (1993) Proc. Natl. Acad. Sci. USA 90, 6586–6590. Boussiotis, V. A., Freeman, G. J., Gray, G., Gribben, J. & Nadler, L. M. (1993) J. Exp. Med. 178, 1753–1763. Hodge, J. W., Abrams, S., Schlom, J. & Kantor, J. A. (1994) Cancer Res. 54, 5552–5555. Zitvogel, L., Tahara, H., Robbins, P. D., Storkus, W. J., Clarke, M. R., Nalesnik, M. A. & Lotze, M. T. (1995) J. Immunol. 155, 1393–1403. Colombo, M. P., Vagliani, M., Spreafico, F., Parenza, M., Chiodini, C., Melani, C. & Stoppacciaro, A. (1996) Cancer Res. 56, 2531–2534. Kubin, M., Kamoun, M. & Trinchieri, G. (1994) J. Exp. Med. 180, 211–222. Coughlin, C. M., Wysocka, M., Kurzawa, H. L., Lee, W. M., Trinchieri, G. & Eck, S. L. (1995) Cancer Res. 55, 4980–4987. Gajewski, T. F., Renauld, J. C., Van Pel, A. & Boon, T. (1995) J. Immunol. 154, 5637–5648. Zitvogel, L., Robbins, P. D., Storkus, W. J., Clarke, M. R., Maeurer, M. J., Campbell, R. L., Davis, C. G., Tahara, H., Schreiber, R. D. & Lotze, M. T. (1996) Eur. J. Immunol. 26, 1335–1341.