Antitumor Activity of Cationic Lipid Complexed with Immunostimulatory ...

doi:10.1006/mthe.2001.0463, available online at http://www.idealibrary.com on IDEAL

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Antitumor Activity of Cationic Lipid Complexed with Immunostimulatory DNA Samantha Rudginsky, William Siders, Laurie Ingram, John Marshall, Ronald Scheule, and Johanne Kaplan* Genzyme Corporation, Framingham, Massachusetts 01701, USA *To whom correspondence and reprint requests should be addressed. Fax: (508) 872-4091. E-mail: [email protected]

We previously reported that treatment of intraperitoneal tumors with complexes of cationic lipid and noncoding plasmid DNA leads to the development of a specific, cytotoxic T-cell response correlating with the rejection of established tumor cells as well as subsequent tumor re-challenge. Here, focusing on an intraperitoneal AB12 mesothelioma model, we show that the anticancer effects of the lipid:DNA complex are associated with DNA containing immunostimulatory CpG motifs. Complexes prepared with cationic lipid and bacterial plasmid DNA, Escherichia coli genomic DNA fragments, or synthetic immunostimulatory CpG oligodeoxynucleotides provided a substantial survival benefit, whereas eukaryotic DNA and methylated bacterial DNA had little or no therapeutic activity. Alternative inflammatory stimuli such as thioglycolate, poly(I:C), and incomplete or complete Freund’s adjuvant failed to reproduce the antitumor activity obtained with the lipid:DNA complex. The innate immune response triggered by lipid:DNA complexes led to the development of a systemic immune response against tumor cells that allowed animals to reject tumors not only at the intraperitoneal treatment site, but also at a distal subcutaneous site. These data demonstrate that immunostimulatory DNA complexed with cationic lipid is a potent inducer of innate and adaptive immune responses against tumor cells and represents a potentially useful tool in the immunotherapy of cancers for which tumor-associated antigens have not been identified. Key words: cationic lipid, DNA, cytokines, CpG motif, innate immunity

INTRODUCTION The application of gene therapy to the treatment of cancer is a promising approach that has met with several limitations. For example, delivery of pro-apoptotic or suicidal transgenes to the majority of cells within a tumor while sparing surrounding normal tissues remains a challenge. By comparison, gene therapies relying on bystander effects or immune stimulatory mechanisms have the theoretical advantage of requiring transduction of only a fraction of the cells within a tumor. Our initial studies focused on such an approach using cationic lipids to deliver plasmids encoding heat shock protein 65 to tumor cells in order to increase their immunogenicity [1]. We, as well as other investigators, observed that complexes of cationic lipid and DNA elicit a potent antitumor response and that this effect could be achieved with noncoding plasmid DNA [1–4]. These observations are consistent with reports on the toxicity and pro-inflammatory activity of complexes of cationic lipid and DNA [5–7]. While the pro-inflammatory activity of cationic lipid:DNA complexes is

MOLECULAR THERAPY Vol. 4, No. 4, October 2001 Copyright © The American Society of Gene Therapy 1525-0016/01 $35.00

detrimental in the context of gene therapy applications seeking to achieve long-term expression of a corrective transgene, this same activity seems to promote the development of innate [1–4] and adaptive [1] immune responses against tumor cells. Mice bearing intraperitoneal (i.p.) AB12 or AC29 mesotheliomas that were treated i.p. with complexes of cationic lipid and noncoding plasmid DNA developed a tumor-specific CD8+ T-cell response that correlated with the destruction of growing tumors and a longterm resistance to further re-challenge with the same tumor cell line [1]. Here, we investigate the applicability of this finding to other tumor models and further characterize the factors involved in the antitumor effect of cationic lipid:DNA complexes.

RESULTS Therapeutic Efficacy of Cationic Lipid:Plasmid DNA Complex in Intraperitoneal Tumor Models We reported previously that multiple intraperitoneal (i.p.) injections of cationic lipid complexed with plasmid DNA

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TABLE 1: Evaluation of treatment with GL67:pNull in various tumor models

Tumor cell line

% Long-term survival after treatment

Strain – species

% Resistance to s.c. tumor re-challenge in surviving animals

AC29 mesotheliomaa

CBA/J mice (H-2k)

> 90%

100%

a

d

20–40%

100%

> 90%

60%

AB12 mesothelioma

Balb/c mice (H-2 )

M3 melanomab

DBA mice (H-2d)

b

b

B16 melanoma

C57BL/6 mice (H-2 )

NuTu 19 ovarian carcinomab b

OVCa ovarian carcinoma a

Results previously published in [1]. b Results not previously published.

0–20%

F344 rat

40–60%

DBIX rat

50%

that the therapeutic benefit of GL67:pNull treatment extends to tumor models other than mouse mesothelioma, but that the level of efficacy achieved is variable and may be affected by factors such as the aggressiveness of tumor growth and the immunogenicity of the tumor cells.

Active Components of GL67:pDNA Complexes Following confirmation of the ther0% apeutic benefit of GL67:pDNA com0% plex in several cancer models, further investigation was initiated to characterize the nature of the active components. We established previously that treatment with cationic lipid or plasmid DNA alone does not provide any substantial survival benefit and that a complex of the two components is required for efficacy [1]. The exact nature of the cationic lipid used to form the complex seemed to have little impact on the therapeutic activity of the complex [1]. To characterize the part played by the DNA component in more detail, DNA from different sources was mixed with GL67 and the therapeutic activity of the resulting complexes was tested. The AB12 mesothelioma model was selected for these comparative studies because it is characterized by aggressive tumor growth that is moderately affected by treatment with complex (40–60% longterm survival), thus allowing for the detection of either an 50%

lacking a transgene (pNull) demonstrated substantial therapeutic activity in two mouse mesothelioma models (Table 1) [1]. Treatment of CBA/J mice bearing i.p. AC29 tumors and BALB/c mice with i.p. AB12 tumors resulted in longterm survival rates of > 90% and 40%, respectively. In both models, the survivors displayed a tumor-specific cytotoxic T lymphocyte (CTL) response and rejected a subsequent subcutaneous (s.c.) tumor cell challenge, indicating that the animals had developed long-lasting systemic immunity against the tumor cells [1]. These findings suggested that administration of cationic lipid complexed with pNull DNA may represent a generalized approach applicable to several cancer types. To assess this possibility, we tested the therapeutic activity of cationic GL67 lipid complexed with pNull plasmid (GL67:pNull) in six different i.p. rodent tumor models (Table 1). Multiple administrations of GL67:pNull were given to mice or rats of different strains bearing syngeneic i.p. tumors of various origins including mesothelioma, melanoma, and ovarian cancer cell lines. GL67:pNull treatment provided a significant but variable survival advantage in all models tested (Table 1). Long-term survival rates ranged from 0 to 20% in the B16 melanoma model to > 90% in the AC29 mesothelioma and M3 melanoma models. The ability of treated animals in a given model to reject subsequent s.c. tumor chal- FIG. 1. Kaplan–Meier survival curves depicting the influence of DNA source on the therapeutic activlenge seemed to be related to the long- ity of GL67:pNull complex. Female BALB/c mice (n = 8/group) were injected i.p. with 5 ⫻ 105 AB12 term survival rate, although the correla- mesothelioma cells on day 0. Injections of cationic lipid GL67 complexed with 100 ␮g DNA in 500 ␮l were given i.p. on days 6, 10, 14, and 18 (arrows). GL67 was complexed with either sssDNA tion was not perfect. For example, in the (square), 5-kb fragments of genomic E. coli DNA (circle), or human DNA (diamond). Control mice AB12 model, complex-treated mice dis- were treated with saline (triangle). Treatment with complexes delivering either sssDNA or human DNA played a long-term survival rate of only did not result in significant increases in survival over a saline control, P > 0.3 for both. In contrast, E. 20–40%, but all surviving animals could coli DNA resulted in a significant increase in survival compared with saline (P < 0.0001). Results are from one experiment which included all of the DNAs in a single study. reject a challenge. These results indicate

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genomic DNA fragments complexed with lipid survived to the end of the study. These results indicate that even DNA of intrinsically higher “potency” requires complexing with a cationic lipid to achieve optimal therapeutic efficacy. Role of Immunostimulatory CpG Motifs in the Antitumor Activity of Prokaryotic DNA Complexed with Cationic Lipid The higher potency of the complex prepared with E. coli genomic DNA fragments, as opposed to sssDNA or human FIG. 2. Therapeutic activity of E. coli DNA fragments administered alone or complexed with GL67. BALB/c mice bearing i.p. AB12 tumors (n = 8) were treated with E. coli genomic DNA alone at doses genomic DNA fragments, suggested the ranging from 50 to 200 ␮g or with 100 ␮g E. coli genomic DNA complexed to cationic lipid GL67 possibility that CpG motifs might be (filled square). Control mice were given saline (open square). Treatments were administered i.p. in 500 involved in the antitumor activity ␮l on days, 6, 10, and 14 (arrows) after the i.p. administration of 5 ⫻ 105 AB12 cells. Mice receiving obtained with prokaryotic DNA. There DNA without cationic lipid showed significant, dose-dependent improvements in survival compared with the saline-treated mice (50 ␮g, P < 0.05; 100 ␮g, P < 0.005; 200 ␮g, P < 0.005). However, the are significant differences in the freenhancement in overall survival was not as pronounced as when the DNA was complexed with cationic quency of CpG dinucleotides in the lipid (P < 0.0001). Results are from a single study due to the practical limitations in producing suffi- genome of bacteria and vertebrates. In cient amounts of endotoxin-free genomic E. coli DNA fragments. bacteria, CpG sequences occur at the expected frequency of 1 out of every 16 bases. This frequency is suppressed in verimpairment or an improvement in efficacy upon manip- tebrate DNA, in which CpG sequences appear at only 10% ulation of the complex. In all studies, AB12 tumor cells of this rate [8]. Most CpG sequences in the vertebrate were administered i.p. and treatment with GL67:DNA genome are also methylated, whereas in bacterial DNA complex was initiated 6 days later. The complex was deliv- they are not [9]. Research over the past several years indicates that unmethylated CpG motifs possess important ered i.p. at 3- to 4-day intervals for a total of 3–4 doses. Treatment with GL67 complexed with supercoiled or immunostimulatory activity and can directly activate cells linearized plasmid (pNull) gave rise to similar levels of of the immune system such as B lymphocytes, monocytes, tumor growth inhibition (P > 0.4 for supercoiled versus macrophages, and dendritic cells [10,11]. In addition, CpG linear), indicating that the conformation of the DNA had DNA induces the release of Th1-type cytokines such as little or no impact on the therapeutic activity of the com- interleukin (IL)-12 and interferon (IFN)-␥, resulting in the plex (data not shown). In contrast, profound differences activation of natural killer (NK) cells [2,3,12]. Methylation in activity were observed when the source of the DNA was of CpG sequences has been shown to eliminate their immunostimulatory activity [3,5,6]. altered (Fig. 1). Escherichia coli genomic DNA fragments To assess the contribution of immunostimulatory CpG complexed with GL67 elicited pronounced antitumor activity, whereas in contrast the activity of human sequences to the antitumor activity of prokaryotic DNA genomic DNA fragments or sheared salmon sperm DNA complexed with GL67, the CpG dinucleotides in E. coli (sssDNA) complexed with GL67 was not statistically dif- genomic DNA were methylated to test whether methylaferent from that of a saline control (P > 0.3 for both treat- tion, and therefore inactivation of immunostimulatory ments versus saline). These results suggested that DNA of CpG sequences, would affect the therapeutic activity of the prokaryotic, as opposed to eukaryotic, origin was required complex. We used SssI methylase to add a CH3 group to for therapeutic efficacy. the cytosine residues present in CpG motifs. Methylation In several studies, the specific activity of E. coli genomic of CpG motifs was confirmed by digesting the DNA with DNA fragments complexed with GL67 seemed to be supe- restriction enzymes sensitive to methylation of the restricrior to that of plasmid DNA. Therefore, the therapeutic tion sites. Treatment of i.p. AB12 tumors with complexes activity of E. coli genomic DNA alone was evaluated in containing methylated as opposed to unmethylated or more detail. Treatment of animals bearing intraperitoneal mock-methylated E. coli genomic DNA resulted in a subAB12 tumors with increasing doses (50–200 ␮g) of E. coli stantial decrease in overall survival (Fig. 3). These results genomic DNA fragments alone (in the absence of cationic support the possibility of a role for unmethylated lipid) resulted in a dose-dependent increase in mean sur- immunostimulatory CpG motifs in the antitumor activity vival time (Fig. 2), but virtually all animals eventually suc- of GL67:bacterial DNA complexes. cumbed to their tumor burden by the end of the study. By To extend this finding, we evaluated synthetic comparison, all of the mice treated with 100 ␮g of E. coli oligodeoxynucleotides (ODNs) containing CpG motifs for


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might exist between the therapeutic activity of a given complex and its ability to induce cytokine production, in particular Th1-type cytokines. Complexes were injected i.p. and the levels of TNF-␣, IFN-␥, IL-6, and IL-12 were measured in both serum and peritoneal lavage at 6 hours after dosing, a time point selected on the basis of previous time-course studies [7]. Elevated levels of TNF-␣ and IL-12 were associated with the i.p. administration of complexes with demonstrated antitumor activity + FIG. 3. Effect of CpG methylation on the potency of E. coli genomic DNA complexed with cationic (GL67 complexed with CpG ODN or E. coli lipid GL67. Groups of eight mice were injected i.p. with 5 ⫻ 105 AB12 mesothelioma tumor cells on genomic DNA fragments) and were not seen day 0 and were treated i.p. on days 4, 7, and 10 (arrows) with GL67 complexed with unmodified in mice injected with nontherapeutic com(square), methylated (circle), or mock-methylated (triangle) E. coli DNA. Control mice were treated plexes (GL67 complexed with sssDNA fragwith saline (diamond). Equivalent increases in survival were observed for the groups treated with – unmodified or mock-methylated DNA (P = 0.42 compared with each other; P < 0.05 compared with ments or CpG ODN). Complexes of + saline). By comparison, methylated DNA showed a significant reduction in therapeutic activity (P < GL67:CpG ODN that produced the highest 0.05 compared with unmethylated DNA). Similar results were obtained in three separate studies. degree of antitumor protection (Fig. 4) also gave rise to the highest serum levels of IL12 and TNF-␣ (Fig. 5). These observations their ability to induce rejection of intraperitoneal AB12 suggest that IL-12 and TNF-␣ may be involved in the develtumors when delivered in a complex with GL67. Both a opment of protective antitumor immunity. No distinct corstimulatory ODN (CpG+) and an irrelevant, nonstimula- relation was seen between levels of IL-6 and IFN-␥ and the tory ODN (CpG–) were tested. The ODNs were synthesized therapeutic potential of the complex injected (data not on a phosphorothioate backbone to reduce nuclease degra- shown). dation [13]. The antitumor activity of CpG+ ODN complexed with GL67 was even greater than that of E. coli Antitumor Activity of Alternative Inflammatory Agents genomic DNA fragments (Fig. 4). By comparison, treatment with CpG– ODN failed to provide any therapeutic As described above, the injection of lipid:pNull complexes advantage over saline. These observations provide further is associated with the production of inflammatory support for the involvement of immunostimulatory CpG cytokines. To determine whether the repeated application of any inflammatory stimulus may be sufficient to produce sequences in the antitumor activity of lipid:DNA coman antitumor response, treatment of i.p. AB12 tumors with plexes. known inflammatory agents was compared with treatment with GL67:pNull complex. Treatment with incomplete Systemic Induction of Cytokines and complete Freund’s adjuvant did seem to increase Following Intraperitoneal Administration of GL67:DNA Complex We showed previously that systemic, long-term immunity is induced following the treatment of intraperitoneal tumors with GL67:pDNA complex and that this immunity is associated with the development of tumor-specific cytotoxic T lymphocytes [1]. The apparent involvement of immunostimulatory CpG sequences in the activity of GL67:DNA complexes introduced the possibility that the induction of cytokine release by CpG dinucleotides may have a role in the development of a cell-mediated immune response. Therefore, the levels of FIG. 4. Role of CpG motifs in the therapeutic activity of GL67:DNA complex. Groups of 10 mice were cytokines present in the peritoneal cav- injected i.p. with 5 ⫻ 105 AB12 mesothelioma tumor cells on day 0 and were treated i.p. on days 6, ity and systemic circulation of animals 10, 14, and 18 with 100 ␮g synthetic ODN that contained (circle) or lacked (triangle) a CpG motif. injected i.p. with complex were evalu- Control mice were treated with saline (diamond) or GL67 complexed with E. coli DNA (square). Results shown are representative of data obtained in three separate studies. ated to determine whether a correlation

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survival over saline treatment, but did not actually result in a statistically significant enhancement (P > 0.05; Fig. 6A). Poly(I:C), which has been reported to stimulate the production of IFN-␥, activate NK cells, and induce maturation of dendritic cells [14], similarly failed to extend survival of AB12 tumor-bearing mice when delivered alone or as a complex with GL67 (Fig. 6B). Repeated i.p. injections of thioglycolate, a known macrophage-activating agent, failed to provide any significant levels of antitumor protection (Fig. 6C). Inhibition of Subcutaneous Tumor Growth Following Local Treatment of Intraperitoneal Tumors The systemic nature of the cytokine response induced by i.p. injection of lipid:E. coli DNA complex (Fig. 5), coupled with our previous observation of resistance to s.c. tumor challenge in complex-treated mice that cured their i.p. tumors, prompted us to test whether local i.p. treatment of i.p. AB12 tumors with complex might lead to eradication of tumor cells seeded at distal sites. For this purpose, mice bearing i.p. AB12 tumors were treated with GL67:E. coli DNA complex and were simultaneously challenged with a s.c. injection of tumor cells. In our hands, the s.c. injection of AB12 tumor cells typically produced a detectable tumor mass by day 10, followed by rapid growth requiring sacrifice of > 90% of the animals by day 30 when tumor size reached ≥ 150 mm2. Control mice treated with saline all died as a result of their i.p. and/or s.c. tumors (Fig. 7). In contrast, most mice (60%) treated with complex were able to reject tumor cells at both sites, resulting in long-term survival. This result indicates that the application of complex to one tumor compartment results in a systemic immune response capable of inhibiting tumor cells present elsewhere in the body.

DISCUSSION These studies confirm and extend our earlier findings on the development of antitumor immunity following treatment with complexes of cationic lipid and noncoding plasmid DNA. Our original observation of tumor growth inhibition following i.p. injection of complex in mice bearing i.p. AB12 or AC29 mesothelioma tumors was found to apply to a variety of other i.p. tumor models, albeit with varying levels of efficacy (Table 1). Further studies in the AB12 mesothelioma model indicated that the antitumor effect of the complex was mediated primarily by unmethylated immunostimulatory CpG sequences present in DNA of bacterial origin. Plasmid DNA alone or cationic lipid alone does not possess any significant antitumor activity; the two components need to be administered as a complex to obtain antitumor protection (Fig. 2) [1]. Although the exact nature and transfection efficiency of the cationic lipid did not seem to substantially affect the therapeutic activity of the complex [1], the nature of the DNA included in the complex was found to be of primary importance.


FIG. 5. Evaluation of cytokine levels in the serum and peritoneal cavity of mice treated i.p. with complexes of GL67 and DNA. Serum and peritoneal lavage samples were collected from groups of mice (n = 4) 6 h after the i.p. injection of GL67 complexed with either stimulatory CpG ODN (filled bars), nonstimulatory CpG ODN (diagonal stripes), E. coli genomic DNA (checker board), sssDNA (vertical stripes), or a saline control (polka-dotted). Samples were analyzed individually by ELISA for the presence of IL-12 (bottom) and TNF-␣ (top). Results shown are the mean ± S.D. of the values obtained with the four samples per group. Similar results were obtained in two separate studies.

The conformation of plasmid DNA (that is, linear or supercoiled) did not seem to affect the activity of the complex, but the inclusion of eukaryotic, as opposed to prokaryotic, DNA essentially abolished the antitumor activity of the complex. We found that i.p. treatment of AB12 tumors with GL67 complexed with DNA of eukaryotic origin, such as sssDNA or human genomic DNA fragments, failed to induce antitumor protection (Fig. 1), whereas complexes of GL67 and E. coli genomic DNA fragments provided a clear survival benefit. Any potential antitumor effects by E. coli DNA contaminants such as endotoxin were ruled out by the observed loss in the activity of complex prepared with DNase-treated E. coli DNA compared with untreated or mock-digested DNA (data not shown).

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A

FIG. 6. Therapeutic activity of nonspecific inflammatory stimuli. Various inflammatory agents were injected i.p. 4–6 d after the i.p. injection of 5 ⫻ 105 AB12 mesothelioma tumor cells and then at 3-d intervals for a total of four doses (n = 10). Complete and incomplete Freund’s adjuvants were given i.p. in 100 ␮l volumes (A), 100 ␮g of poly(I:C) was injected with or without cationic lipid GL67 in a 500 ␮l volume (B), and 4% thioglycolate was injected in a 1 ml volume (C). In each instance, control groups included saline-treated mice and mice treated with GL67:pNull complex. None of the inflammatory agents extended survival to the same degree as GL67:pNull. Results are from a single study with each agent.

B

the role of lipid in the complex was to protect the DNA from degradation by nucleases and/or to facilitate entry of the DNA into cells, as immune activation by bacterial DNA appears to require cellular uptake [1,20,21]. A rapid induction of cytokine release following i.v., i.p., or intratracheal administration of lipid:DNA complex was previously reported [2–4,7,22] and was also observed here. Several cytokines, including TNF-␣, IL-12, IL-6, and IFN-␥, were detected in the peritoneal cavity and serum of mice injected i.p. with complex (Fig. 5). At 6 hours postinjection, a time point previously determined to be optimal for cytokine detection [7], a correlation was seen between the antitumor potential of a given complex and its ability to induce elevated levels of TNF-␣ and IL-12. For example, complexes of GL67 and CpG+ ODN, which induced the highest level of antitumor protection, also produced the largest increase in circulating levels of TNF-␣ and IL-12, whereas only background levels of these cytokines were detected in mice injected with GL67 complexed with sssDNA or CpG– ODN. In a related finding [7], systemic administration of a CpG-rich plasmid (pCF1-CAT) complexed with cationic lipid induced cytokine release, whereas injection of the same plasmid without lipid failed to induce cytokine production. TNF-␣, IFN-␥, and IL-12 participate in the activation of NK cells, macrophages, and CTLs [2], and it may be that they have an integral role in the development of the antitumor response elicited by lipid:DNA complexes. The induction of IL-6 and IFN-␥ was also detected in some complex-treated mice, but there was no obvious correlation between the therapeutic activity of a given complex and the level of induction of these cytokines. However, the study focused on a single time point, and it is possible that more extensive studies of the kinetics of these treatments may have revealed a stronger correlation between antitumor protection and induction of IL-6 and IFN-␥. In particular, the induction of IFN-␥ production following administration of lipid:DNA complex [2–4,6,7] was reported to be required for rejection of MCA-205 lung tumors in mice injected i.v. with cationic lipid:DNA complex [3]. Overall, our cytokine data indicate that the i.p. injection of cationic lipid complexed with immunostimulatory DNA leads to the rapid and systemic induction of pro-inflammatory cytokines with known immunoactivating and antitumor properties, which may participate in the development of the observed antitumor response.

C

The immunostimulatory properties of bacterial DNA are well-documented and have been attributed to the increased frequency and hypomethylated state of CpG motifs in bacterial DNA compared with eukaryotic DNA [15,16]. Bacterial DNA and synthetic ODNs containing immunostimulatory CpG motifs stimulate macrophages, B lymphocytes, and dendritic cells and activate NK cells through the induction of IL-12 and TNF-␣ [10,17–19]. A role for immunostimulatory CpG motifs in the antitumor effect of GL67:E. coli DNA complex was supported by the observation of reduced therapeutic activity upon methylation of CpG dinucleotides in the bacterial DNA (Fig. 3). Furthermore, treatment of i.p. AB12 tumors with GL67 complexed with a synthetic ODN containing a stimulatory CpG motif elicited a strong antitumor response, whereas GL67 complexed with a nonstimulatory ODN failed to provide any protection from tumor growth (Fig. 4). The induction of an antitumor immune response upon intravenous administration of lipid:DNA complexes has been reported by other investigators, who also observed a reduction in activity upon methylation of the DNA and consequently proposed a role for immunostimulatory CpG motifs in the antitumor effect [3,4]. It was postulated that

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types. Our data (Table 1) support this interpretation, as the greatest rate of tumor rejection and long-term survival was obtained with tumors that possess some inherent immunogenicity (for example, AC29 mesothelioma and M3 melanoma), compared with nonimmunogenic tumor types (such as B16 melanoma) for which treatment with complex succeeded in delaying tumor growth rather than causing tumor rejection. Based on these observations, one could envisage that lipid:DNA FIG. 7. Effect of local treatment with complex on the growth of tumor cells at distal sites. GL67:E. treatment might be most useful as a standcoli DNA complex was used to treat AB12 mesothelioma tumors at two anatomical sites. Peritoneal alone therapy against more immunogenic tumors were seeded on day 0 in all groups and s.c. tumors were seeded in a subset of animals on day 8 (square and triangle). GL67:E. coli DNA complex (square and circle) or saline (triangle or dia- tumor types, whereas combination with mond) treatments were administered i.p. on days 3, 6, 10, and 14. In mice bearing i.p. tumors only, other anti-cancer agent(s) may be required treatment with complex was efficacious in 70% of animals, whereas all saline-treated mice died by for effectiveness against more aggressive, day 34. In mice bearing both i.p. and s.c. tumors, 3 of 10 saline-treated mice (triangles) developed nonimmunogenic tumors. measurable s.c. tumors before they were found moribund and the rest succumbed to their peritoneal Finally, the systemic nature of the protumors. None of the complex-treated mice that received both peritoneal and s.c. injections of AB12 cells (square) developed measurable s.c. tumors and 60% rejected their i.p. tumors. Similar results inflammatory response elicited by i.p. were obtained in the M3 melanoma tumor model, in which complex delivered i.p. led to the erad- administration of lipid:DNA complex, in ication of both peritoneal tumors and distal lung metastases (W.S., unpublished data). conjunction with our previous observation of a specific CTL response following treatment with complex, suggests that local delivery of lipid:DNA into a tumor-bearing compartment The ability of lipid:DNA complexes to induce promight give rise to a systemic antitumor response capable of inflammatory cytokines may not entirely account for their eradicating tumor cells growing at distal sites. This hypothantitumor activity, as repeated i.p. administration of other esis was supported by a study showing that i.p. delivery of nonspecific inflammatory stimuli (such as thioglycolate, lipid:DNA complex to mice bearing both i.p. and s.c. AB12 poly(I:C), and complete or incomplete Freund’s adjuvant) tumors led to the rejection of tumor cells at both sites and failed to duplicate the antitumor effect of complex. allowed for long-term survival in most animals (Fig. 7). Our Similarly, i.v. administration of LPS or poly(I:C) did not results indicate that complexes of cationic lipid and lead to the rejection of pulmonary metastases that was immunostimulatory DNA represent an attractive treatment observed following i.v. injection of cationic lipid:DNA complex [3]. Additional immunological mechanisms are modality that may provide a therapeutic benefit against a apparently triggered by the injection of lipid:DNA com- broad range of tumor types. plex, which ultimately lead to tumor rejection and development of specific antitumor immunity. It has been MATERIALS AND METHODS reported that CpG DNA seems to be recognized by a TollPlasmids. All plasmids were constructed at Genzyme Corporation like receptor (TLR9) different from the Toll-like receptors (Framingham, MA). The 5-kb pNull plasmid lacks a transgene, but contains involved in the recognition of bacterial components like a CMV promoter, a BGH poly(A) tail, and the kanamycin resistance gene LPS (TLR4) or peptidoglycan (TLR2) from organisms such for purification purposes. The plasmid DNA was prepared by bacterial feras the mycobacteria present in complete Freund’s adjuvant mentation followed by ultrafiltration and sequential column chromatog[23]. Signaling through different receptor pathways by CpG raphy purification [24]. Fermentation was carried out in a 15-liter Chemap fermenter using HCD medium (Genzyme Corp., Framingham, MA) with DNA versus bacterial cell wall components may be involved 100 ␮g/ml kanamycin at 37⬚C for 24 h. The resulting DNA was predomiin the observed differences in antitumoral activity. nantly supercoiled with spectrophotometric A260/ A280 ratios of 1.75–2. The We demonstrated previously in the AB12 and AC29 plasmid DNA was free of detectable levels of RNA. In addition, it contained + mesothelioma models that both NK cells and CD8 T cells < 10 ␮g of protein, 10 ␮g of chromosomal DNA, and 5 endotoxin units per mg of plasmid DNA. Endotoxin levels were determined using the LAL assay are required for the antitumor activity elicited by complex and that animals that successfully reject their tumors (Bio Whittaker, Walkersville, MD) [8]. Linearization of the plasmid DNA demonstrate a specific CTL response [1]. These findings was carried out by digestion with BamHI (Gibco BRL, Rockville, MD). Plasmids used were supercoiled unless otherwise indicated. suggest that treatment with lipid:DNA complex nonGenomic DNA. Genomic human and E. coli DNA were obtained from specifically triggers a potent innate immune response, Sigma (St. Louis, MO). The genomic DNA was hydrated by shaking which in turn promotes the development of a specific anti- overnight in sterile water (Baxter, Deerfield, IL) at room temperature. The genomic DNA was then digested with EcoRI (Gibco BRL) to produce fragtumor response. According to this scenario, treatment with complex would be expected to be more effective against ments of ~ 5 kb. Three aliquots of 5000 U of EcoRI were added to the immunogenic compared with nonimmunogenic tumor genomic DNA (approximately 1 mg/ml) daily for 3 d while the genomic DNA was gently shaken at 37⬚C. Endotoxin was then removed from the


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genomic DNA preparations using Detoxigel (Pierce, Rockford, IL) as described [25]. Briefly, genomic DNA was incubated with the gel slurry at 4⬚C for 20 min and the gel slurry was then removed via sequential centrifugation steps. The resulting genomic DNA preparation contained less then 5 endotoxin units per mg as determined by the LAL assay (BioWhittaker). The sssDNA was obtained from 5⬘-3⬘, Inc. (Boulder, CO).

Tumor oligodeoxynucleotides. Synthetic ODNs containing stimulatory CpG sequence (5⬘-TCCATGACGTTCCTGACGTT-3⬘) or nonstimulatory CpG sequence (5⬘-GCGGCGGGCGGCGCGCGCCC-3⬘) with a phosphorothioate backbone were obtained from Operon Technologies, Inc. (Alameda, CA). The lyophilized ODNs were reconstituted with sterile water before complexing with cationic lipid.

DNA methylation. EcoRI-digested E. coli genomic DNA fragments (15–17 mg) were methylated in a 15 ml reaction containing 1⫻ NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol) supplemented with 160 ␮M S-adenosylmethionine, and 1 unit SssI methylase per 25 ␮g DNA for 18 h at 37⬚C. After 18 h, an additional aliquot of S-adenosylmethionine was added and the digestion was continued for another 3 h. Mock-methylated DNA was subjected to the same treatment with the omission of the SssI methylase. After digestion, the DNA was purified by means of phenol/chloroform and chloroform extractions. Methylation was confirmed by digestion with the methylation-sensitive HaeIII enzyme, which does not cut methylated GGmCC sequences, and the methylation-insensitive HhaI enzyme (data not shown).

Cytokine assays. Levels of IL-6, IL-12, interferon-␥, and TNF-␣ present in serum or peritoneal lavage samples were analyzed using commercial ELISA kits from R&D Systems (Minneapolis, MN) according to the manufacturer’s instructions.

Alternative immunostimulatory agents. Polyinosinic-polycytidylic acid (poly[I] • poly[C]; Sigma) was injected alone or complexed with GL67 lipid as described below. Thioglycolate Medium Brewer Modified (4.05% thioglycolate solution; Becton Dickenson, Franklin Lakes, NJ) was administered as a 1 ml intraperitoneal (i.p.) injection [26]. Complete and incomplete Freund’s adjuvants (Gibco BRL) were injected i.p. in a 100 ␮l volume. Cationic lipid and formation of complex. Cationic lipids were produced at Genzyme Corporation. The cationic lipid GL67 (N4-spermine cholesterylcarbamate) was prepared by small molecule synthesis as described [24]. The neutral co-lipid dioleoylphosphatidylethanolamine (DOPE; Avanti Polar Lipids, Inc., Alabaster, AL) was mixed with GL67 in chloroform at a molar ratio of 1:2, GL67:DOPE. The lipid mixture was transferred to sterile vials and the chloroform removed by evaporation. The lipid was hydrated before use with the addition of sterile water (Baxter) for 10 min followed by 2 min of vigorous vortexing. For complex formation, the lipid and nucleic acid were each warmed in a 30⬚C water bath and then mixed by adding the lipid to the nucleic acid at a molar ratio of 1:4 cationic lipid:nucleic acid (for example, 150:600 ␮M). Complexing was then allowed to proceed at 30⬚C for 15 min before diluting with an equal volume of 1.8% sodium chloride solution to provide a final concentration of 0.9% sodium chloride. Complexes were administered within 30 min of preparation. Cell lines. The murine mesothelioma cell lines AB12 and AC29 were obtained from Bruce Robinson (Queen Elizabeth II Medical Center, Perth, Australia) through Steven Albelda (University of Pennsylvania, Philladelphia, PA). Both cell lines were induced by the i.p. implantation of asbestos fibers into BALB/c or CBA/J mice [27]. The M3 melanoma cell line derived from the DBA mouse strain was obtained from Stressgen Biotechnology (Victoria, British Columbia) and the B16-F10 melanoma cell line syngeneic to C57BL/6 mice was obtained from the National Cancer Institute (Bethesda, MD). The DBIX rat ovarian cancer cell line, OV-Ca, was obtained from the German Cancer Center (Heidelberg, Germany) through Stressgen Biotechnology. The NuTu-19 ovarian cancer cell line of Fischer rat origin was obtained from Thomas Hamilton (Fox Chase Cancer, Philadelphia, PA). Tumor implantation and treatment. To establish AB12 or AC29 tumors, 5 ⫻ 105 cells were injected into the peritoneal space of 7- to 9-week-old female BALB/c or CBA/J mice (Taconic Laboratories, Germantown, NY), respectively. Similarly, M3 or B16-F10 peritoneal tumors were established in 7- to 9-week-old female DBA or C57BL/6 mice (Taconic Laboratories), respectively, by injecting 2 ⫻ 105 cells. Finally, peritoneal tumors were generated by injection of 5 ⫻ 105 OV-Ca cells into the peritoneal cavity of female DBIX rats weighing approximately 125 g (Charles River Laboratories, Wilmington, MA). NuTu-19 cells were given as an i.p. injection of 1 ⫻ 106 cells to female Fischer rats weighing approximately 90 g. We administered 3–4 treatments i.p. with cationic lipid:DNA complex or DNA alone at 3- to 4-d intervals starting 3–6 d after the administration of tumor cells.

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Statistical analysis. Kaplan-Meier survival curves were analyzed with the Mantel-Cox Log-rank test. Cytokine data are expressed as the mean value from four individual mice ± standard error.

ACKNOWLEDGMENTS We thank our Genzyme colleagues Nick Wan for the production and purification of the plasmid DNA; Mikaela Keynes and David Harris, for the GL67 formulations; the Laboratory Animal Research Group for their thorough monitoring of the survival studies; and members of both the Gene Transfer Research Group and Gene Therapy Immunology Group for discussions. RECEIVED FOR PUBLICATION MAY 1; ACCEPTED AUGUST 3, 2001.

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