Immune Cell-Mediated Antitumor Activities of GD2- Targeted Liposomal c-myb Antisense...

Immune Cell–Mediated Antitumor Activities of GD2-Targeted Liposomal c-myb Antisense OligonucleotidesContaining CpG Motifs

Chiara Brignole, Fabio Pastorino, Danilo Marimpietri, Gabriella Pagnan,Angela Pistorio, Theresa M. Allen, Vito Pistoia, Mirco Ponzoni

Background: Expression of the c-myb proto-oncogene inneuroblastoma, the most common extracranial solid tumorof infancy, is linked with cell proliferation and differentia-tion. Neuroblastoma can be selectively targeted via mono-clonal antibodies against the disialoganglioside (GD2) tumor-associated antigen. Liposomes coated with anti-GD2antibodies (targeted liposomes) and entrapping a c-mybantisense oligonucleotide have antitumor activity. Becauseantisense oligonucleotides containing CpG motifs can stim-ulate immune responses, we evaluated the effect of CpG-containing c-myb antisense oligonucleotides encapsulatedwithin targeted liposomes. Methods: Antisense (myb-as) andscrambled (myb-scr) control oligonucleotides with CpG mo-tifs were encapsulated within GD2-targeted and non-targeted liposomes. Two murine (nude and SCID-bg) xeno-graft models of neuroblastoma were established. Mice(groups of 10) were injected intravenously with various oli-gonucleotide and liposome formulations, and life span, long-term survival, immune cell activation, and cytokine releasewere measured over time. Results: Tumor-bearing mice in-jected with targeted liposome-CpG-myb-as or targeted lipo-some-CpG-myb-scr lived longer than mice in any othergroup, although long-term survival (i.e., more than 120 days)was obtained only in mice injected with targeted liposome-CpG-myb-as. Splenocytes isolated from mice injected withtargeted liposome-CpG-myb-as contained activated macro-phages, B cells, and natural killer (NK) cells, but only acti-vated NK cells were associated with antitumor cytotoxicactivity. In vivo immune cell activation was accompanied bythe time-dependent increases in plasma levels of the cyto-kines interleukin 12 (IL-12; maximum level reached by 2hours) and interferon gamma (IFN-�; maximum levelreached by 18 hours) and was dependent on the oligonucle-otide CpG motif. Ablation of macrophages or NK cells re-sulted in a loss of in vivo antitumor activity. Conclusion:Immune cell activation, involving the time-dependent acti-vation of macrophages and NK cells, contributes to theantitumor activity of targeted liposome-CpG-myb-as againstneuroblastoma and could improve the effectiveness of anti-tumor targeted liposomes. [J Natl Cancer Inst 2004;96:1171–80]

Neuroblastoma, a neoplasm derived from the sympatheticnervous system, is the most common extracranial solid tumor ofchildhood (1,2). Despite advances in treatment strategies, long-term survival for patients with advanced-stage disease is poor(3,4).

The identification of cancer-associated molecules and/orgenes holds promise for the development of novel therapeuticstrategies that selectively target tumor cells. Among these strat-

egies, antisense oligonucleotides can be used to decrease tumor-associated gene expression, resulting in specific anticancer ef-fects and minimal damage to normal tissues. In addition to theirdirect effects on gene expression, antisense oligonucleotides canhave antitumor effects via indirect, immune-stimulatory mech-anisms, if they are designed to contain unmethylated cytosine–guanine (CpG) motifs. These motifs can induce cytokine secre-tion and stimulate innate immune responses (5–7). Thesefeatures make CpG-containing antisense oligonucleotides poten-tially useful as immune adjuvants.

Although antisense oligonucleotides, with or without CpGmotifs, show promise as therapeutic agents for direct or indirecttumor cell killing, there are a number of problems associatedwith their clinical use. First, antisense oligonucleotides are sen-sitive to cleavage by ubiquitous nuclease enzymes in vivo (8)—aproblem that can be circumvented by the synthesis of chemicallymodified antisense oligonucleotides. However, structural modi-fications to antisense oligonucleotides, e.g., the addition of phos-phorothioate groups to increase their resistance to nuclease deg-radation, may compromise their activities or lead to their bindingto nonspecific DNA sequences (9,10). Second, delivery of freeantisense oligonucleotides is often limited by their low bloodstability, cellular uptake, and specificity. These problems can bereduced by incorporating the antisense oligonucleotides intolipid vesicles or liposomes. Indeed, liposome encapsulation ofantisense oligonucleotides specific for the raf oncogene led toincreased levels of antisense oligonucleotides in plasma andtissues and improved antitumor effects relative to the free oligo-nucleotides (11). Liposomes have been found to facilitate thedelivery of antisense oligonucleotides targeting the Bcl-2,MDR1, or c-myc genes in vivo (12–15).

Sterically stabilized (i.e., containing polyethylene glycol[PEG] grafted onto the liposome exterior) immunoliposomesthat have cell surface–directed antibodies coupled to the PEGterminus can selectively deliver drugs and diagnostic agents totarget cells [reviewed in (16)]. Principles similar to those used inthe therapeutic applications of sterically stabilized immunolipo-somal drugs can be applied to selectively deliver antisenseoligonucleotides to target cells in vivo.

Affiliations of authors: Laboratory of Oncology (CB, FP, DM, GP, VP, MP),and Epidemiology and Biostatistics Service (AP), G. Gaslini Children’s Hospi-tal, Genoa, Italy; Department of Pharmacology, University of Alberta, Edmon-ton, AB, Canada (TMA).

Correspondence to: Mirco Ponzoni, PhD, Differentiation Therapy Unit, Lab-oratory of Oncology, G. Gaslini Children’s Hospital, Largo G. Gaslini 5, 16147Genoa, Italy (e-mail: [email protected]).

See “Notes” following “References.”

DOI: 10.1093/jnci/djh221Journal of the National Cancer Institute, Vol. 96, No. 15, © Oxford UniversityPress 2004, all rights reserved.

Journal of the National Cancer Institute, Vol. 96, No. 15, August 4, 2004 ARTICLES 1171

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The disialoganglioside GD2 is a tumor-associated antigen thathas appeal for use in targeting applications involving liposome-encapsulated drugs or liposome-encapsulated antisense oligo-nucleotides because it is widely expressed on cancer cells ofneuronal origin. Moreover, ligand or antibody binding to GD2

leads to the internalization of the receptor. In normal tissues,GD2 is expressed at low levels and only in the cerebellum andperipheral nerves.

The c-myb proto-oncogene is the best characterized memberof the myb family of transcription factor genes. Expression ofc-myb has been detected in several solid tumors of differentorigin, including neuroblastoma, and is linked to cell prolifera-tion and/or differentiation (17,18). Recently, we demonstratedthat c-myb antisense oligonucleotide entrapped in GD2-targetedliposomes inhibited neuroblastoma growth in vitro by specifi-cally reducing c-myb protein expression (19,20). Here, we in-vestigated the direct (i.e., anti-c-myb–mediated) and the indirect(i.e., CpG-mediated) antitumor effects of GD2-targeted c-myb-antisense–containing liposomes in xenograft mouse models ofhuman neuroblastoma that mimic the natural clinical course ofthis disease (21,22).

MATERIALS AND METHODS

Chemicals and Monoclonal Antibodies

Hydrogenated soy phosphatidylcholine, cholesterol, 1,2-distearoylglycero-3-phosphatidylethanolamine-N-polyethyleneglycol-2000 (DSPE-PEG), and 1,2-dioleoyl-3- trimethylammo-nium propane were purchased from Avanti Polar Lipids (Ala-baster, AL). A derivative of DSPE-PEG with a maleimide groupat the distal terminus of the polyethylene glycol chain wassynthesized by Shearwater Polymers (Huntsville, AL). [3H]cho-lesteryl hexadecyl ether was purchased from DuPont NEN (Bos-ton, MA). The hybridoma cell line 14.G2a, which secretes amurine immunoglobulin G2a (IgG2a) specific for the GD2 an-tigen, was a generous gift of R. A. Reisfeld (The ScrippsInstitute, La Jolla, CA). All of the biochemical- and molecularbiology–grade reagents were obtained from Sigma Chemical(St. Louis, MO).

Oligonucleotides

We used a 24-base phosphorothioate c-myb antisense oligo-nucleotide that contains CpG sequences, which is referred tohereafter as CpG-myb-as. The antisense sequence is comple-mentary to codons 2–9 of the human c-myb mRNA and has thefollowing sequence: 5�-TATGCTGTGCCGGGGTCTTCGGGC-3�. We also used a scrambled sequence, 5�-TTTGCAGTCCTGGGGTCGTGGGCC-3�, which contains a CpG motif(CpG-myb-scr), as a control oligonucleotide. Both CpG-myb-asand CpG-myb-scr were provided by Inex Pharmaceuticals(Burnaby, BC, Canada). A second scrambled sequence, 5�-TGTGCAGTCCTTGGGCTGTGGGCC-3�, lacking the CpGmotifs (myb-scr) was purchased from TIB Molbiol (Genoa,Italy) and was used as an additional control sequence.

Liposome Preparation

Oligonucleotides were encapsulated within coated cationicliposomes by using the method of Stuart et al. (13), with ourslight modification (19). Fab� fragments of the anti-GD2 mono-

clonal antibody and of an isotype-matched control monoclonalantibody (isotype-matched Fab) were coupled to the terminus ofmaleimide-containing DSPE-PEG on the external surface of theliposome (targeted liposome) by a procedure that we recentlydescribed (21,23).

In Vivo Therapeutic Studies

HTLA-230 cells were a gift from E. Bogenmann (Chil-dren’s Hospital Los Angeles, Los Angeles, CA). HTLA-230cells were isolated from a patient with metastatic stage IVneuroblastoma. These cells exhibit high levels of N-myc geneamplification and of GD2 expression and grow in athymicnude mice (21,24). This cell line was grown in completemedium, which consisted of Dulbecco’s Modified Eagle Me-dium, supplemented with 10% fetal bovine serum (Sigma), 50IU/mL sodium penicillin G, 50 �g/mL streptomycin sulphate,and 2 mM L-glutamine, as previously described (19). Five-week-old female athymic (nude) mice and SCID-bg (C.B-17/IcrHsd-scid-bg) mice were purchased from Harlan Laborato-ries (Harlan Italy-S. Pietro al Natisone, Udine, Italy) andhoused under specific pathogen-free conditions. Mice re-ceived intravenous injections via the tail vein of 3.5 � 106

HTLA-230 tumor cells in HEPES-buffered saline (21). After4 hours, mice were randomly assigned to groups of 10, andeach mouse received the first intravenous oligonucleotide orcontrol treatment. Treatments were administered once a day,4 days per week (a single course), with a 3-day intervalbetween courses, for a total of 2 weeks. Each treatmentconsisted of 50 �g of oligonucleotide per mouse. Oligonu-cleotides were administered in various formulations: CpG-myb-as, liposome-CpG-myb-as, liposome-CpG-myb-scr, li-posome-myb-scr, targeted liposome-CpG-myb-as, andtargeted liposome-CpG-myb-scr. Mice assigned to the controlgroup received HEPES-buffered saline, CpG-myb-as plusanti-GD2 Fab� fragments, empty targeted liposome, and iso-type-matched Fab�–targeted liposome-CpG-myb-scr.

Therapeutic studies were also performed in mice depletedof macrophages, which was accomplished via administrationof liposomes containing dichloromethylene bisphosphonate(clodronate; Sigma) (25), as previously described (26). Todeplete the macrophages, mice were given intravenous injec-tions of clodronate-containing liposomes 2 days before re-ceiving the HTLA-230 tumor cells and beginning treatment(25,26).

The body weight and general physical status of the mice wererecorded daily, and mice were killed when signs of poor healthbecame evident. All experiments involving animals have beenreviewed and approved by the licensing and ethical committeeof the National Cancer Research Institute and by the ItalianMinistry of Health. All in vivo experiments were repeated atleast twice, with each set of experiments yielding similar results.

Studies on Immune Stimulation

Stimulation of immunocompetent cells was investigatedboth in vitro and ex vivo. Briefly, for the in vitro studies,spleens from nude mice that had received neither tumor cellsnor oligonucleotide treatments were collected and reducedmechanically into single-cell suspensions. Splenocytes werethen cultured in complete medium in the presence of CpG-myb-as or targeted liposome-CpG-myb-as at an oligonucleo-

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tide concentration of 20 �g/mL. Splenocytes harvested atvarious time points after incubation with the oligonucleotideswere processed for flow cytometry to determine the pheno-type of the cells. To detect specific cell populations, we usedanti-CD11b (M1/70) antibodies for monocytes, anti-CD19(1D3) antibodies for B lymphocytes, anti-NK 1.1 (PK136)antibodies for NK cells, anti-GR-1 (RB6-8C5) antibodies forgranulocytes, and anti-CD11c (HL3) antibodies for myeloiddendritic cells. All antibodies were conjugated with phyco-erythrin and were purchased from BD Biosciences (SanDiego, CA). Cell activation was assessed by double-stainingB lymphocytes, monocytes, NK cells, granulocytes, and my-eloid dendritic cells with a fluorescein–isothiocyanate-conjugated anti-CD69 monoclonal antibody (BD Bio-sciences), which detects an early activation antigen, inconjunction with the cell-specific monoclonal antibodies. Inall experiments, splenocytes were incubated first with Fc-Block (BD Biosciences) to block Fc receptors and subse-quently with test monoclonal antibodies or isotype-matchedmonoclonal antibodes of irrelevant specificity, both conju-gated with the same fluorochromes. Cells were analyzed on aFACScan flow cytometer using Cell Quest software (BectonDickinson, Mountain View, CA). A total of 1 � 104 eventswere collected for each sample.

The threshold for positive expression was based on the max-imum staining obtained with the isotype-matched monoclonalantibody (control antibodies) used at the same concentration asthe test antibody. If fewer than 1% of the cells stained positivewith control antibodies, then the cell population was considerednegative for the marker. If cells labeled with test antibody werebrighter than those stained with isotype-matched control anti-body, then the cell population was considered positive for themarker.

For the ex vivo studies, 1 day after receiving the tumor cellinoculation, nude mice received a single intravenous injection ofeither liposome-CpG-myb-as, targeted liposome-CpG-myb-as,or CpG-myb-as. Each injection delivered 80 �g of oligonucle-otide per mouse. HEPES-buffered saline, in a volume equivalentto that used for the injection of the oligonucleotide, was injectedinto control mice. At different times after injection, mice (threeper group) were anesthetized with ketamine, blood was collectedas described below, and the mice were killed. Spleens wereharvested and splenocytes were isolated, as described above, andpooled within the same group. At the same time points, a sampleof peripheral blood was collected through the retro-orbital sinusfrom each of the same three mice. Peripheral blood was col-lected in a tube containing heparin (2 IU/mL; Parke-Davis,Milan, Italy). The blood was centrifuged (at 500g for 10 minutesat 4 °C) and pooled within groups, and the plasma was isolatedand stored at –20 °C. The collected peripheral blood cells andsplenocytes were processed and stained for CD69 expression,which was assessed by flow cytometry. Plasma concentrations ofthe cytokines interleukin 12 (IL-12), interferon gamma (IFN- ),interleukin 1 beta (IL-1�), and tumor necrosis factor alpha(TNF-�) were measured with commercially available enzyme-linked immunosorbent assay kits, according to the manufactur-er’s recommended protocols. Kits for IL-12, IFN- , and TNF-�were purchased from Bender MedSystems Diagnostics (Vienna,Austria), and the kit for IL-1� was purchased from BiosourceInternational (Camarillo, CA).

Cytotoxicity Assays

Cytotoxicity was measured by the use of a standard 51Cr-release assay (27). Briefly, three mice per group were giveninjections, at 80 �g of oligonucleotide per mouse, of CpG-myb-as, liposome-CpG-myb-as, or targeted liposome-CpG-myb-as.Control mice received injections of HEPES-buffered saline.Mice were killed at different times after injection, and spleno-cytes and peritoneal macrophages were collected from eachmouse and pooled within the same treatment group.

The peritoneal macrophages (approximately 1 � 105

cells/mL to 2 � 105 cells/mL) were cultured overnight in com-plete medium. Splenocytes were cultured overnight in Dulbec-co’s Modified Eagle Medium in the absence or presence ofrecombinant IL-2 (1000 IU/mL; Proleukin Chiron, Emeryville,CA). The HTLA-230 neuroblastoma target cells were labeled for2 hours with Na2

51CrO4 (100 �Ci/1 � 106 cells) (ICN Biomedi-cals, Asse, Belgium). Target cells were then incubated for 6hours at 37 °C with splenocytes or peritoneal macrophages aseffector cells. The effector : target cell ratio was 100 : 1 forsplenocytes and 10 : 1 for macrophages. Supernatants were col-lected, and the amount of radioactivity released into the super-natants was counted with a gamma counter (Cobra 5002; Can-berra Packard, Meriden, CT). The percentage of specific targetcell lysis was calculated using the formula [(Exp – S)/(M – S)] �100%, in which Exp is the observed released 51Cr value, S is thespontaneously released 51Cr value, and M is the maximumreleased 51Cr value.

Statistical Methods

Results are expressed as mean � 95% confidence intervals(CIs). All in vitro data are from at least three independentexperiments. All in vivo experiments were performed at leasttwice. Survival curves were constructed by using the Kaplan–Meier method, and the log-rank test was used to compare thecurves. Bonferroni’s adjustment was applied as a correction formultiple comparisons to explore post hoc differences betweenpairs of groups. A P value of less than .05 was consideredstatistically significant. Hazard ratios of death were calculatedwith 95% confidence intervals. Statistical analyses were per-formed using Statistica (StatSoft, Tulsa, OK) and Stata, release7.0 software (Stata, College Station, TX).

RESULTS

In Vivo Effects of Targeted Liposomal myb AntisenseOligonucleotides

To evaluate the antitumor efficacy of different liposomeformulations of myb antisense oligonucleotides in a murinemodel of human neuroblastoma, initial experiments were per-formed in a biologically and clinically relevant xenograft modelalready established in our laboratory (21,22). Mice injected withthe targeted liposome-CpG-myb-as lived longer (mean � 4months) than control mice injected with buffer (mean � 1.5months) (hazard ratio [HR] of death � 0.31, 95% CI � 0.12 to0.80; P�.001) (Fig. 1) or than control mice injected with thetargeted liposome-CpG-myb-scr (HR � 0.55, 95% CI � 0.22 to1.39; P � .004). However, a statistically significant, althoughsmaller, increase in life span was also seen in mice injected withthe targeted liposome-CpG-myb-scr compared with mice in-



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jected with buffer (HR � 0.57, 95% CI � 0.24 to 1.37; P�.001),mice injected with CpG-myb-as (HR � 0.58, 95% CI � 0.24 to1.40; P�.001), mice injected with the combination of CpG-myb-as plus the Fab� fragment of anti-GD2 (HR � 0.59, 95% CI� 0.24 to 1.41; P�.001), or mice injected with the emptytargeted liposome (HR � 0.62, 95% CI � 0.26 to 1.49;P�.001), suggesting that our data were not exclusively the resultof an antisense effect or of stimulation of the immune system bythe Fab� targeting moiety, as expected from our previous results(23,27).

Mice injected with liposome-CpG-myb-as (HR � 0.76, 95%CI � 0.32 to 1.83; P � .003) or liposome-CpG-myb-scr livedlonger than control mice, although not as long as mice injectedwith the targeted liposome-CpG-myb-scr (HR � 0.75, 95% CI� 0.31 to 1.80; P � .009). The life span of mice injected withliposome-CpG-myb-as or liposome-CpG-myb-scr was similar tothat of mice injected with liposome-CpG-myb-scr targeted via anonspecific Fab� fragment (NS-targeted liposome-CpG-myb-scr) (Fig. 1). This observation is in agreement with our previousfinding (19) that ligand-mediated specific targeting of antisenseoligonucleotide liposomes increases the antitumor effectsagainst neuroblastoma cells. However, because similar anti-tumor effects were obtained with the scrambled and antisenseoligonucleotides in the nontargeted liposome formulations, asecond mechanism, in addition to antisense-mediated loss of theoncogene protein, was required to explain the results. Becauseboth the myb-as and the myb-scr oligonucleotides containedCpG motifs and because CpG-containing antisense oligonucle-otides can activate innate immune responses (7), we hypothe-sized that our liposomes may work by an additional nonspecificmechanism involving activation of innate immune responses bythe CpG motifs.

In Vitro and In Vivo Administration of TargetedLiposome-CpG-myb-as and Immune Activation

To assess the potential involvement of CpG motifs in theinduction of an innate immune response by our liposome for-mulations, we evaluated the expression of the CD69 antigen, anearly marker of cellular activation, both ex vivo and in vitro, onvarious immunologically relevant cell types (28,29). The ex vivostudies were performed on splenocytes and peripheral bloodcells collected from tumor-bearing nude mice that had receivedtargeted or non-targeted liposome-CpG-myb-as or CpG-myb-as.Flow cytometric analysis of splenocytes demonstrated that in-jection of targeted liposome-CpG-myb-as resulted in a rapid(within 2 hours) increase of CD69 antigen expression on mono-cytes, B cells, and NK cells (Fig. 2, A). Liposome-CpG-myb-asdid not increase CD69 expression on any splenocyte population(data not shown). However, injection of liposome-CpG-myb-asactivated monocytes from peripheral blood, although to a lesserextent and on a different time scale than injection of targetedliposome-CpG-myb-as (Fig. 2, B). Compared with splenocytesor blood cells from mice injected with HEPES buffer, spleno-cytes or blood cells from mice injected with CpG-myb-as orCpG-myb-as plus the Fab� fragment of anti-GD2 antibodies wereonly marginally activated, if at all (data not shown).

We next evaluated whether the in vitro stimulation of spleencells could mimic the ex vivo stimulation. Splenocytes werecollected from tumor-free nude mice, cultured in a completemedium, and treated in vitro with CpG-myb-as, liposome-CpG-myb-as, targeted liposome-CpG-myb-as, or NS-targetedliposome-CpG-myb-scr at an oligonucleotide concentration of20 �g/mL. After splenocytes were treated with the targetedliposome-CpG-myb-as, CD69 expression increased in a time-dependent manner for each of the different cell populations, i.e.,B cells, monocytes, NK cells, granulocytes, and dendritic cells.Maximum CD69 expression was detected after 18 hours (Fig. 3).CD69 expression was only marginally increased in splenocytestreated with liposome-CpG-myb-as or CpG-myb-as (data notshown). Thus, intravenous injection of targeted liposome-CpG-myb-as induced a marked immune response in neuroblastoma-bearing mice.

Intravenous Injection of Targeted Liposome-myb-as andCytokine Secretion

Because cytokine secretion is another marker of immune-cellactivation, we next assessed the effect of intravenous injectionsof liposome-CpG-myb-as on cytokine release over time intumor-bearing mice. Levels of IL-12, IFN- , IL-1�, and TNF-�cytokines were higher in mice injected with targeted liposome-CpG-myb-as than in mice injected with liposome-CpG-myb-as(Fig. 4). Cytokine levels in mice injected with CpG-myb-as werecomparable to cytokine levels in control mice injected withHEPES buffer. After mice were injected with targeted liposome-CpG-myb-as, the maximum IL-12 level was achieved within 2hours and was approximately eightfold (4425 pg/mL, 95% CI �4312 to 4538 pg/mL) higher than the level in control mice (570pg/mL, 95% CI � 485 to 655 pg/mL) injected with buffer or inmice injected with CpG-myb-as. The levels of IL-12 in miceinjected with non-targeted liposomes were lower than the levelsin mice injected with targeted liposomes, but the kinetics wassimilar, reaching a peak within 2 hours and declining rapidlythereafter (Fig. 4, A).

Fig. 1. Survival of neuroblastoma-bearing nude mice after injection with lipo-somes containing oligonucleotides. Nude mice were injected intravenously with3.5 � 106 HTLA-230 neuroblastoma cells and randomly assigned to groups of10 mice. After 4 hours, each mouse received 50 �g of oligonucleotide, eitherfree or encapsulated in either targeted or non-targeted liposomes. Control micereceived HEPES-buffered saline. Survival of mice was monitored daily. Fortargeted liposome-CpG-myb-as versus control, hazard ratio of death � 0.31[95% confidence interval � 0.12 to 0.80; P�.001].



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Levels of IFN- in mice injected with targeted liposome-CpG-myb-as began to increase after 8 hours and were approxi-mately sevenfold (256 pg/mL, 95% CI � 233 to 279 pg/mL)higher than levels in control mice (34 pg/mL, 95% CI � 31 to37 pg/mL) after 18 hours (Fig. 4, B). The levels of IFN- werelower in mice injected with liposome-CpG-myb-as than in miceinjected with targeted liposome-CpG-myb-as. These results areconsistent with the hypothesis that formulations containing CpGsequences initially stimulate production of IL-12 by macro-phages, which may, in turn, activate NK cells to secrete IFN- .Mice injected with targeted liposome-CpG-myb-as sustainedhigh levels of IL-1� and TNF-� compared with mice injected

with HEPES-buffer or with CpG-myb-as. Mice injected withliposome-CpG-myb-as had lower levels of IL-1� and TNF-�than mice injected with targeted liposome-CpG-myb-as (Fig. 4,C and D). Thus, intravenous injection of targeted liposome-CpG-myb-as elicited a strong systemic secretion of immuno-stimulatory cytokines in neuroblastoma tumor–bearing mice.

Intravenous Injection of Targeted Liposome-CpG-myb-asand NK Cell Cytotoxic Activity

Because oligonucleotide-containing liposomes appear towork, in part, by stimulating the innate immune system, wedesigned experiments to identify the immunologic effectors oftumor cell killing. In an in vitro 51Cr-release assay, peritonealmacrophages collected at different times after tumor-bearingnude mice were injected with either targeted or non-targetedliposome-CpG-myb-as did not lyse the HTLA-230 neuroblas-toma target cell line (data not shown). By contrast, peritonealmacrophages isolated from tumor-bearing mice and incubatedwith the anti-GD2 monoclonal antibody mediated antibody-dependent cellular cytotoxicity activity against the target cells(data not shown), as expected (27).

We repeated the in vitro 51Cr-release assay with splenocytesfrom tumor-bearing mice injected with either targeted or non-targeted liposome-CpG-myb-as as effector cells. Splenocytesfrom mice injected with targeted liposome-CpG-myb-as specif-ically lysed the neuroblastoma HTLA-230 target cells in a time-dependent manner (Fig. 5). Cytotoxic activity was increasedwhen the splenocytes were incubated with IL-2 to promote NKcell activation (30,31). Splenocytes from mice injected withtargeted liposome-CpG-myb-as were more effective thansplenocytes from mice injected with liposome-CpG-myb-as at

Fig. 2. Expression of the activation antigen CD69 ex vivo on immune cells.Splenocytes (A) and peripheral blood cells (B) were collected fromneuroblastoma-bearing nude mice at different times after injection of c-mybantisense oligonucleotides containing CpG motifs encapsulated in GD2-targetedor non-targeted liposomes. Cell populations and expression of CD69 weredetermined by two-color flow cytometry using phycoerythrin-conjugated mono-clonal antibodies to identify cell type and a fluorescein–isothiocyanate-conjugated anti-CD69 monoclonal antibody. Monocytes were identified withanti-CD11b antibodies, B cells with anti-CD19 antibodies, and NK cells withanti-NK 1.1 antibodies. A) The cell type and percentage of cells expressingCD69 was determined for splenocytes isolated from mice injected with targetedliposome-CpG-myb-as. B) Peripheral blood monocytes expressing CD69 iso-lated from mice injected with targeted liposome-CpG-myb-as or non-targetedliposome-CpG-myb-as. Control mice received HEPES-buffered saline. For eachtime point, the splenocytes or peripheral blood cells from three mice were pooledand analyzed, and the results were expressed as mean with 95% confidenceintervals. Each experiment was repeated three times, with similar results.

Fig. 3. Expression of the activation antigen CD69 in vitro on immune cells.Splenocytes were collected from tumor-free nude mice and cultured (approxi-mately 5 � 106 cells/mL) for different times in the presence of c-myb antisenseoligonucleotides containing CpG motifs encapsulated in either targeted lipo-somes or control buffer (HEPES-buffered saline). Cell populations and expres-sion of CD69 were determined by two-color flow cytometry usingphycoerythrin-conjugated monoclonal antibodies to identify cell type and afluorescein–isothiocyanate-conjugated anti-CD69 monoclonal antibody. Mono-cytes were identified with anti-CD11b antibodies, B cells with anti-CD19 anti-bodies, NK cells with anti-NK 1.1 antibodies, dendritic cells with anti-CD11cantibodies, and granulocytes with anti-Gr-1 antibodies. For each time point, theresults are expressed as mean (95% confidence intervals) of three differentexperiments.



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mediating the lysis of the neuroblastoma HTLA-230 target cells(Fig. 5), confirming the in vivo results (Fig. 1). Thus, intravenousadministration of targeted liposome-CpG-myb-as elicited astrong functional activation of NK cells. Because the experi-ments were done with cells from nude mice, which lack T cells,the functional activation of the NK cells is T-cell-independent.

Effects of Depletion of Macrophages or NKCells on Antitumor Efficacy ofOligonucleotide-Containing Liposomes

To gain additional insights into the in vivo relevance of ourobservations, therapeutic experiments were conducted in micechemically depleted of macrophages with liposomes containingclodronate (26). Macrophage depletion led to a complete loss ofantitumor efficacy in neuroblastoma-bearing mice injected withliposome-CpG-myb-as (Fig. 6, A). As a further control, we alsoused a liposome formulation in which the scrambled oligo-nucleotide sequence lacked the CpG motifs. Survival curvesshowed a complete loss of antitumor effects in macrophage-depleted mice treated with liposome-myb-scr (data not shown).

With the final aim of distinguishing the specific antisenseactivity of our oligonucleotide-containing liposomes from thenonspecific CpG-mediated immune responses, we conductedtherapeutic experiments using SCID-bg mice, which lack NKcells. These mice were chosen because NK cells were theonly effector cells identified in the in vitro tumor cytotoxicityassay (Fig. 5). Neuroblastoma-bearing SCID-bg mice injectedwith targeted liposome-CpG-myb-as lived statistically signif-icantly longer than control mice (HR of death � 0.63, 95% CI

� 0.25 to 1.59; P � .002) or mice injected with targetedliposome-CpG-myb-scr (Fig. 6, B), although not as long asnude mice injected with targeted liposome-CpG-myb-as. Inthis experiment, 120 days after receiving the tumor cells, 20%of the mice injected with targeted liposome-CpG-myb-aswere still alive compared with none of the mice in the othertreatment groups. These data demonstrate that the therapeuticresults with targeted liposome-CpG-myb-as obtained in ourneuroblastoma tumor nude mouse model were likely attrib-utable to both the decreased expression of the c-myb proto-oncogene (19) and to the nonspecific stimulation of the hostimmune system mediated by the CpG motifs.

DISCUSSION

This study demonstrated that the systemic administration ofCpG-containing antisense oligonucleotides, directed against thec-myb oncogene, to mice bearing human neuroblastoma inducedantitumor effects leading to long-term survival only when theantisense oligonucleotides were encapsulated in coated cationicliposomes specifically targeted to the neuroblastoma surfaceantigen GD2 that internalizes after binding its ligand. Increasedlife span, but not long-term survival, was also obtained in miceinjected with targeted liposomes of CpG-containing scrambledc-myb oligonucleotides and, to a lesser degree, in mice injectedwith non-targeted liposomes of CpG-containing antisense orscrambled oligonucleotides. These findings support a dual mech-anism of action of the antisense oligonucleotide liposomes:direct inhibition of cell growth (resulting from decreased c-myb

Fig. 4. Plasma cytokine levels innude mice injected intravenouslywith HEPES-buffered saline(control), c-myb antisense oligo-nucleotides containing CpG mo-tifs (CpG-myb-as), or c-mybantisense oligonucleotides con-taining CpG motifs encapsulatedin GD2-targeted (targeted lipo-some-CpG-myb-as) or non-tar-geted liposomes (liposome-CpG-myb-as). Plasma levels ofinterleukin 12 (IL-12) (A), inter-feron gamma (IFN- ) (B), inter-leukin 1 beta (IL-1�) (C), andtumor necrosis factor alpha(TNF-�) (D) were determined byusing commercially available en-zyme-linked immunosorbent as-says and the results are expressedas picograms of cytokine per mil-liliter of plasma. Each data pointrepresents the mean cytokinelevel (95% confidence intervals)from three mice. The experimentwas repeated three times, withsimilar results.



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proto-oncogene expression) and indirect CpG-dependent im-mune stimulation (resulting from NK cell-mediated lysis oftumor cells).

Therapeutic effects of antisense oligonucleotides werethought to arise primarily from molecular complementarity(10,32), which leads to inhibition of gene expression in asequence specific manner. Recently, activities unrelated toantisense effects, such as immune stimulation, have beenreported (7,33). This immunostimulatory activity is stronglydependent on the presence of unmethylated CpG motifs (5).In contrast to bacterial DNA, which contains a large numberof unmethylated CpG motifs, mammalian DNA containsmostly methylated CpG motifs (34). Immune effector cellsseem to have evolved pattern recognition receptors that, bybinding the CpG sequences, evoke protective immune re-sponses (35). Specific recognition of CpG motifs occurs viabinding to the TLR9 receptor, which belongs to the super-family of Toll-like receptors (35). It has recently been dem-onstrated that the intravenous injection of lipid–DNA com-plexes into tumor-bearing mice induces antitumor activity(28,36) and that the encapsulation of CpG-containing oligo-nucleotides in lipidic particles greatly increases their immu-nostimulatory effects (37). Immune stimulation mediated byCpG sequences may therefore have a role in anticancertherapy.

Targeted lipid-based carriers, such as coated cationic lipo-somes, may solve many of the problems associated with the invivo use of antisense oligonucleotides (13,14,19,38,39). Ourprevious studies (19,21) showed that the encapsulation of eithermyb-as or myc-as oligonucleotides within GD2-targeted coatedcationic liposomes improved their efficacy against neuroecto-derma-derived tumor cells. In fact, treatment of neuroblastomacells in vitro, and of melanoma cells in vitro and in vivo, with

GD2-targeted coated cationic liposomes containing myb-as ormyc-as oligonucleotides, respectively, led to an inhibition oftumor cell proliferation via the specific loss of the expression ofproliferative proteins (19,21,40).

In this study, we investigated the antitumor effects of GD2-targeted liposomes containing CpG-myb-as oligonucleotides ina biologically and clinically relevant experimental mouse modelof human neuroblastoma (21,22). We found that the systemicadministration of GD2-targeted liposomes containing CpG-myb-as to neuroblastoma-bearing mice led to long-term sur-vival. Some of the observed effects were the result of CpG-mediated immune stimulation, because we demonstrated that theantitumor activity was associated with both targeted and non-targeted liposomes containing oligonucleotides with CpG mo-

Fig. 5. Cytotoxic activity in spleen cells isolated from tumor-bearing nude miceafter intravenous administration of c-myb antisense oligonucleotides containingCpG motifs (CpG-myb-as) or c-myb antisense oligonucleotides containing CpGmotifs encapsulated in GD2-targeted (targeted liposome-CpG-myb-as) or non-targeted liposomes (liposome-CpG-myb-as). Control mice were injected withHEPES-buffered saline (control). Splenocytes, cultured in the absence or pres-ence of recombinant interleukin 2 (IL-2), were used as effector cells in 51Cr-release assays against HTLA-230 neuroblastoma target cells. The effector :target cell ratio was 100 : 1. The results are expressed as means of three differentexperiments with 95% confidence intervals.

Fig. 6. Effect of depleting macrophages or natural killer (NK) cells on antitumoractivity associated with oligonucleotide-containing liposomes in murine xeno-graft models of neuroblastoma. A) Nude mice (groups of 10) were injected withliposomes containing clodronate 2 days before they were injected with HTLA-230 neuroblastoma cells. After 4 hours, the mice were injected with c-mybantisense oligonucleotides containing CpG motifs encapsulated in liposomes(liposome-CpG-myb-as). B) SCID-bg mice (groups of 10) were injected withHTLA-230 neuroblastoma cells and injected 4 hours later with GD2-targetedliposome-CpG-myb-as (targeted liposome-CpG-myb-as) or targeted liposome-CpG-myb-scr. Control mice received HEPES-buffered saline. Survival of micewas monitored daily. For targeted liposome-CpG-myb-as versus control, hazardratio of death � 0.63 (95% confidence interval � 0.25 to 1.59; P � .002).



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tifs, in agreement with previous findings (41). Further evidencederives from the observation that no increase in life span wasfound for neuroblastoma-bearing mice treated with a non-targeted liposome containing oligonucleotides without CpGmotifs.

We speculate that long-term survival in neuroblastoma-bearing mice was related to several factors working in coop-eration: long circulation times of the liposomes (required forlocalization to the neuroblastoma), selective targeting of theliposomes to the neuroblastoma tumor cells via GD2 (requiredto reduce nonspecific uptake of the liposomes), and CpG-mediated immune stimulation enhancing the specific anti-sense activity of myb-as oligonucleotides (required for loss ofc-myb expression). Thus, on the basis of our in vivo resultsand previous studies (28,41), we hypothesize that macro-phages initiate a cascade that ends with activated NK cellskilling neuroblastoma cells. This hypothesis is supported bythe observations that abrogation of macrophages by lipo-somes containing clodronate (26) abolished the antitumoractivity associated with liposome-CpG-myb-as. The criticalinvolvement of NK cells was demonstrated by the total loss ofantitumor activity in experiments in which targeted liposomesentrapping a scrambled CpG-containing oligonucleotide wereadministered to neuroblastoma-bearing NK-cell– depletedSCID-bg mice. However, in the SCID-bg mouse model, treat-ment with targeted liposomes containing CpG-myb antisenseoligonucleotide still resulted in long-term survival for 20% ofthe mice, suggesting that the specific inhibition of c-mybplays a pivotal, but partial, role in its antitumor efficacy.These experiments allowed us to separate the antitumor ac-tivity associated with the delivery of the CpG sequence tomacrophages from that associated with the specific loss ofc-myb expression.

The mechanisms underlying the immunogenicity of our lipo-some formulations were investigated both in vitro and ex vivo.Targeted and non-targeted liposomes increased CD69 expres-sion on macrophages from peripheral blood. However, only thetargeted liposomes rapidly induced activation of immune cellsfrom splenocytes. Compared with targeted liposomes, non-targeted liposomes have longer circulation times and lowersplenic uptake, which may explain the difference in effectsbetween the two formulations (13). Targeted liposomes showedsome accumulation in the spleen (15,20), which may give rise torapid activation of splenocytes. The intravenous injection ofoligonucleotide-containing liposomes led to the release of anumber of immunostimulatory cytokines into plasma. The ki-netics of secretion are consistent with the idea that macrophagesare the first mediators of the immune response with an earlyrelease of IL-12 (peak at 2 hours), which triggers, in turn, therelease of IFN- by NK cells (peak at 18 hours). This finding isin agreement with previous results (42). However, in our murinemodel, the contribution of other cell types to the release ofIFN- and IL-12 cannot be ruled out. Indeed, IL-12-activatedmacrophages and B cells are induced to secrete IFN- (43,44).The increased, sustained levels of TNF-� could cooperate withIL-12 to induce lytic activity of NK cells, confirming previousresults, which demonstrated that both IL-12 and TNF-� arerequired for the induction of NK cell lytic activity by CpG-containing oligonucleotides (42,45,46). Likewise, IL-1� can beinvolved early in the generation of lymphokine-activated killercells (47). Finally, the hypothesis that NK cells play a pivotal

role in killing neuroblastoma cells was supported by the obser-vation that splenocytes collected from mice injected with ouroligonucleotide-containing liposomes were able to lyse neuro-blastoma cells.

In conclusion, the encapsulation of antisense oligonucleotidesin targeted liposomes substantially increased their therapeuticactivity against neuroblastoma cells by a direct mechanism in-volving the loss of expression of the c-myb proto-oncogene andby an indirect mechanism involving antisense oligonucleotidesthat contain immune-stimulatory CpG motifs. Because manymelanomas express high levels of GD2, have altered expressionof c-myb, and are growth inhibited by c-myb antisense oligo-nucleotides, there may be a potential role for targeted c-mybantisense therapy in the treatment of melanoma. However, as anote of caution, targeted liposomes appear to lose their efficacywhen they are used to treat established advanced solid tumors(48,49), likely because the binding site barrier restricts penetra-tion of liposomes into the tumor (50,51). We contend thattargeted liposome-CpG-myb-as deserves clinical evaluation asan adjuvant therapy for advanced-stage neuroblastoma diseaseor for disease resulting from incomplete surgery or early micro-metastatic lesions.

A final comment addresses the potential relevance of theexperimental therapeutic approach described herein in atumor-bearing host with an intact immune system. It may beenvisaged that the availability of a large amount of IFN- ,consequent to CpG stimulation of the innate immune system,has a strong impact on the tumor cells themselves. In humanneuroblastoma, IFN- can sensitize tumor cells to apoptosis(52) or induce their differentiation, especially in conjunctionwith TNF-� (53); IFN- alternatively could increase expres-sion of the class I major histocompatibility complex antigenon the cell surface (54), thus allowing killing of neuroblas-toma cells by tumor-specific cytotoxic T lymphocytes. In thisscenario, it is apparent that administration of CpG-containingoligonucleotides to a tumor-bearing immunocompetent hostwould give rise to multiple opportunities for the host immunesystem to destroy cancer cells. The contribution of eachmechanism to tumor cell killing in this setting remains to beestablished.

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NOTES

C. Brignole and F. Pastorino contributed equally to the work.Supported by Fondazione Italiana per la lotta al Neuroblastoma and Associa-

zione Italiana Ricerca Cancro (AIRC).

We thank R. A. Reisfeld for providing us with the hybridoma 14.G2a, whichsecretes a murine anti-GD2 monoclonal IgG2a antibody; M. Hope for providing uswith the myb-antisense and myb-scrambled oligonucleotides; L. Moretta and A.Mantovani for helpful discussion; F. Morandi for expert technical help; M. Cilli forexpert technical assistance; and C. Bernardini for editing. F. Pastorino is a recipientof a Fondazione Italiana per la Lotta al Neuroblastoma fellowship. C. Brignole is arecipient of a Fondazione Italiana Ricerca Cancro (FIRC) fellowship.

Manuscript received December 29, 2003; revised May 21, 2004; acceptedJune 8, 2004.



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