prolonged survival of thymoma-bearing mice after...

(CANCER RESEARCH 52. 6287-6291, November 15. 1992]

Prolonged Survival of Thymoma-bearing Mice after Vaccination with a SolubleProtein Antigen Entrapped in Liposomes: A Model Study1

2,3Fan Zhou,2 Barry T. Rouse, and Leaf Huang

Departments of Biochemistry [F. Z., L. H.J and Microbiology [B. T. R.Â¡and Cell, Molecular, ana Developmental Biological Program [F. Z., B. T. R., L. HJ,University of Tennessee, Knoxville, TN 37996-0840

ABSTRACT

EG7-OVA cells are mouse thymoma EL4 cells stably transfectedwith the complementary DNA of chicken m Â¡illumini(OVA) and thusexpress OVA epitopes as a unique antigen. Cytotoxic T lymphocytesspecific to OVA can be elicited by immunization of mice with OVAosmotically loaded into syngeneic splenocytes or entrapped in liposomes. Cytotoxic T lymphocytes thus induced can specifically cytolysethe EG7-OVA cells in vitro in an antigen-specific and major histocom-patibility complex-restricted manner. In the present study, we haveexamined in this model system whether immunization with liposomalOVA can protect mice against tumors induced by EG7-OVA cells.Vaccination with OVA either entrapped in liposomes or osmoticallyloaded in the syngeneic splenocytes prolonged the survival of micewhich had been challenged with EG7-OVA cells, but not those micechallenged with the parent 114 cells. The antitumor effect was attributed to the induced OVA-specific cytotoxic T lymphocyte activity, sinceother forms of acquired immunity such as interaction of tumor cells withspecific antibody could not be detected. Our results demonstrate thatimmunization with antigen incorporated in liposomes could be a usefulmeans of inducing a protective antitumor response.

INTRODUCTION

The role of different components of the host immune systemin the suppression of tumor progression has been under investigation for several decades. Observations that emphasize theimportance of NK4 cells, macrophages, lymphokine-activatedkiller cells, tumor-infiltrating lymphocytes, and CTL have beenreported by different groups of investigators (1-5). Controversial findings have revealed the complexity of tumor immunology. With extensive study in preclinical animal models, it hasbeen realized that different subpopulations of effectors make acritical contribution to antitumor immunity in particular cases.NK cells could be the major immune mechanism for destroyingtumors that express low levels of MHC class I molecules ontheir surface (6). Tumors with normal expression of class Imolecules, such as EL4 thymoma, are sensitive to cytolysis byCTL (7). Transfer of tumor-specific CTL that are expanded invitro is very effective in prolonging the survival of tumor-bearing animals (8). Although CD4+ CTL, lymphokine-activated

killer cells, and macrophages are indeed actively involved intumor elimination, CD8+ CTL are responsible for the direct

eradication of tumor cells in many malignant diseases (9).

Received 5/29/92; accepted 9/4/92.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by Grants CA 24553. Al 29893, and AI 24762 fromthe NIH.

2 Current address: Department of Pharmacology, University of PittsburghSchool of Medicine, Pittsburgh. PA 15261.

3 To whom requests for reprints should be addressed, at Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.

4 The abbreviations used are: NK, natural killer; CTL, cytotoxic T lymphocyte(s);MHC, major histocompatibility complex; OVA, ovalbumin; PBS. phosphate-buffered saline: NGPE. /V-glutarylphosphatidylethanolamine; HEPES, 4-<2-hydroxyeth-yl)-l-piperazineethanesulfonic acid; DMEM. Dulbecco's modified Eagle's medium;ELISA, enzyme-linked immunosorbent assay.

Therefore, it is conceivable that protection might be gained bypreferential induction of tumor-specific CTL using appropriate

vaccination.However, development of tumor vaccines has met with only

limited success. One of the obstacles is the low efficiency ofeliciting tumor antigen-specific CTL via inoculation with purified antigens or tumor lysates (10, 11). It is well known thatpriming of a CTL response to a specific antigen requires thepresence of the antigen in the cytosol of antigen-presentingcells. Only the endogenous, cytosolically located, antigen isusually available for the class I pathway (12). Exogenous antigen processed via the endocytic route is presented with class IImolecules, leading to antibody production (13).

Several approaches to introduce antigens into the class Ipathway have been reported. Proteins or peptides conjugatedwith lipophilic compounds (14) or formulated with immuno-

stimulating complex (15) are capable of inducing a CTL response. We have employed the pH-sensitive liposome approachto deliver OVA to the cytosolic compartment of EL4 thymomacells (16). The EL4 cells which are sensitized with liposomalOVA can be recognized by specific CTL. Specific lysis of thesensitized EL4 cells is comparable to that of EG7-OVA cells,which are transfected to endogenously produce OVA with anH-2Kb-restricted CTL epitope (17, 18). Recently, we have ex

tended the liposome delivery research to in vivo CTL inductionand demonstrated that liposome formulations can also primean OVA-specific and class I-restricted CTL response (19).

Since EG7-OVA cells differ from the parent EL4 cells byexpressing a unique antigen, i.e., OVA, we have tested in thismodel system whether the OVA-specific CTL response elicitedby liposomal formulations can protect mice which are challenged with EG7-OVA cells or the parent EL4 cells. The resultshave shed some light on the role of liposomal delivery of potential tumor-specific antigens in protection against tumors.

MATERIALS AND METHODS

Chemicals. Dioleoyl phosphatidylethanolamine, dioleoyl phospha-tidylcholine, l,2-dioleoyl-i/i-3-succinylglycerol, and phosphatidylserinewere purchased from Avanti Polar Lipids (Birmingham, AL). OVA andcholesterol were obtained from Sigma Chemical Co. (St. Louis, MO).NGPE was synthesized as described elsewhere (20). The purity of theproduct was determined by thin layer chromatography before usefor protein modification. Na25'CrO4 was from ICN BiomÃ©dical.Inc.(Costa Mesa, CA). '-"I-Anti-mouse IgG and I25I-OVA were prepared

by iodination with lodoBeads (Pierce, Rockford, IL).Cell Lines and Animals. EL4 is a thymoma cell line of H-2h haplo-

type and P815 is a mastoma cell line of H-2d haplotype. The EG7-OVA

cell line, an EL4 cell line stably transfected with OVA complementaryDNA, was a kind gift from Dr. M. Bevan (Howard Hughes MedicalInstitute, Seattle, WA) and was maintained in the presence of G418(400 Mg/rnl).All cells were cultured in DMEM supplemented with 10%fetal calf serum, 10 HIMi.-glutamine, 100 Mg/ml streptomycin, and 100units/ml penicillin, at 37Â°Cin 7% CO2. Female C57BL/6 mice (5-6

weeks of age) were provided by HarÃanSpraguc Dawley, Inc. (Indianapolis, IN) and were used I week after their arrival at our animalfacility. All animal experiments were conducted in accordance with the

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ANTITUMOR IMMUNE RESPONSE

"Guide for the Care and Use of Laboratory Animals," as proposed by

the Committee on Care of Laboratory Animals Resource Commissionon Life Science-National Research Council.

Liposome Preparation. Liposome 1, which was composed of dio-leoyl phosphatidylethanolamine and l,2-dioleoyl-in-3-succinylglycerol(1:1, mole ratio), and liposome 2, which was composed of dioleoylphosphatidylcholine, phosphatidylserine, and cholesterol (5:2:3, moleratio), were prepared by a freeze-thaw method described previously(17). Briefly, a thin lipid film of 10 nmol of lipid containing a traceamount of hexadecyl pHJcholestanyl ether was hydrated in 250 M' ofHEPES-sucrose buffer (5 HIMHEPES, 0.29 Msucrose, pH 8.0) at 4Â°C

for 72 h. During hydration, the lipid suspension was sonicated and thepH of the suspension was adjusted, if necessary, to 8.5-9.0 with l NNaOH. An OVA solution containing a trace amount of I25I-OVA was

then added to a final concentration of 25 mg/ml. The mixture wassubjected to three cycles of freeze-thaw. Following brief sonication,OVA encapsulated in liposomes was separated from unentrapped freeOVA by chromatography using a Bio-Gel A 1.5 Mcolumn which wasequilibrated with PBS (pH 8.0).

Osmotic Loading of OVA Antigen in Splenocytes. Splenocytes wereprepared from naive C57BL/6 mice and osmotically loaded with OVAas described by Okada and Rechsteiner (21). Briefly, 1 x IO8 RBC-

depleted Splenocytes were suspended in 1 ml of hypertonic OVA medium (20 mg/ml OVA, 0.5 M sucrose, 10%, w/v, polyethylene glycol1000, 10 HIMHEPES, in RPMI 1640 medium, pH 7.0) at 37Â°Cfor 10

min. The cell suspension was then osmotically shocked by addition of15 volumes of hypotonie medium (60% Hanks' balanced salt solution,40% distilled H2O) and incubated at 37Â°Cfor 3 min. The Splenocytes

were then washed twice with cold RPMI 1640 medium and X-irradiated(2000 rad) before use for immunization.

Cytotoxic T Lymphocyte Induction and Assay. Mice were immunized i.v. with OVA entrapped in liposomes (100 Mg/mouse) or insplenocytes (25 Mg/mouse) or free in PBS (100 Mg/mouse). Mice usedfor secondary responses were boosted with OVA entrapped in liposome1 one week after the primary immunization with OVA in splenocytes.After 7-10 days of injection or booster, the spleens were asÃ©pticaIlyremoved and the RBC-depleted splenocytes were prepared. The primary' CTL population was restimulated with X-irradiated (20,000 rad)EG7-OVA cells in limiting dilution medium (50% RPMI 1640 mediumand 50% NCTC 109 medium, supplemented with 10% fetal calf serum,10 imi L-glutamine, 100 Mg/ml streptomycin, 100 units/ml penicillin,and 50 MM2-mercaptoethanol) at 37Â°Cfor 5 days and then used as

effectors. Target cells (EL4, EG7-OVA, and P815) were radioactivelylabeled with 5lCr and added to serially diluted effectors in 96-wellmicroplates. After a 4-h incubation at 37"C, 100 n\ of supernatant were

harvested and counted in a gamma counter. The specific lysis of targetcells was determined as follows:

Specific lysis (%) =experimental release - spontaneous release

total release - spontaneous release

Protective Effect of the OVA-specific CTL Induction in Vivo.Groups of five mice were immunized i.v. with OVA encapsulated inliposomes or splenocytes or free in PBS. OVA antigen administered permouse was 100 pg for the liposomal preparation and 25 Mg for thesplenocyte preparation. Seven days after the injection, mice were challenged with EL4 cells or EG7-OVA cells by i.v. injection of 5 x IO6

cells/mouse. The mice were observed for up to 50 days to determine themean survival time of each group.

Determination by ELISA of OVA-specific IgG Production. The production of OVA-specific antibody was measured by ELISA. Ninety-six-well microplates were coated with OVA (25 Mg/ml) in bicarbonatebuffer, pH 9.6, at 4Â°Covernight. After extensive washing with PBS-

Tween, 200 M'of blocking buffer (10% goat serum in PBS-Tween) wereapplied to each well and incubated at 37Â°Cfor 2 h. The blocking buffer

was removed before the addition of 100 ml of mouse serum in threedifferent dilutions (50:1, 100:1, and 200:1 ). The reaction was allowed toproceed at 37Â°Cfor 2 h. The microplates were washed and 100 n\ of

horseradish peroxidase-conjugated anti-mouse IgG (1:1000 dilution)

were added to each well. The microplates were washed again afterincubation at 37Â°Cfor 2 h. One hundred n\ of substrate solution were

then added and incubated at room temperature for 5-15 min. Thereaction was terminated by addition of 50 n\ of 2 NH2SO4 to each well.The absorbance at 405 nm was determined with an automatic micro-plate reader (model EL 301; Bio-Tek Instruments Inc., Winooski, VT),using appropriate reagent blanks. ELISA units were calculated by multiplying the corrected absorbance value at 405 nm by the reciprocal ofthe serum dilution.

Binding of Anti-OVA Antibody to the Tumor Cells. EL4 cellswith surface-associated OVA (EL4-OVA) were prepared with the following procedure. First, OVA was chemically modified with a phos-pholipid, NGPE, by the method of Holmberg et al. (20). Briefly, NGPEwas activated to the /V-hydroxysuccinimide ester with l-ethyl-3-(3-dim-ethylaminopropyl)carbodiimide (Sigma) in 2-(iV-morpholino)ethane-sulfonic acid buffer, pH 5.0, containing 10-20 HIMoctylglucoside. After10 min at room temperature, the pH of the reaction solution wasbrought to 7.5 with l NNaOH. I25I-OVA in 10 m\i HEPES buffer, pH7.4, was added and the incubation was continued at 4Â°Cfor 12 h. To

remove the detergent, the resulting preparation was dialyzed against 4liters of PBS, pH 7.4, at 4Â°Covernight. The dialyzed NGPE-OVA wasthen added to an EL4 cell suspension (250 MgNGPE-OVA/IO6 EL4cells) in serum-free DMEM and incubated at 4Â°Cfor 1 h with interval

shaking. After washing with DMEM, the cell-associated radioactivitywas quantitated and the incorporation of modified OVA into the EL4cells was calculated.

Binding of anti-OVA to tumor cells was determined as follows. Al-iquots of cells (EG7-OVA, EL4, or EL4-OVA) were suspended inDMEM, in 15-ml centrifuge tubes. Cells were incubated with 10-folddiluted mouse anti-OVA serum at 4Â°Cfor 1 h. After washing by cen-trifugation at 1000 x g for 5 min, 131I-anti-mouse IgG was added to thecell suspension and incubated at 4Â°Cfor 1 h. Cells were washed again

and gamma counted. The percentage of radioactivity associated withcells was determined.

Complement-mediated Cell Lysis. EL4 cells (1 x 106/sample),which had been previously radioactively labeled with Na^'CrO.Â», wereincubated with mouse anti-OVA serum (1:10 dilution) or sheep ant iThy-1 serum (1:1000 dilution) at 4Â°Cfor 1 h. After thorough washing,cells were incubated with 1:20 diluted rabbit complement at 37Â°Cfor

1 h. Cells were then pelleted and the supernatants were removed forgamma counting. Specific release of 5lCr from cells was calculated bythe same formula used for calculation of CTL-mediated lysis.

Statistical Analysis. Data from the antibody binding and complement-mediated cytolysis assays were analyzed using the Student t test.The mouse survival curves were drawn according to the Kaplan-Meierproduct limit method and the mean survival of grouped mice was compared by analysis of variance using StatView II software.

RESULTS

Prolongation of Survival of EG7-OVA-bearing Mice by Immunization with OVA Encapsulated in Membranous Vehicles.Without any pretreatment, most of the mice inoculated witheither EG7-OVA or EL4 tumors died within 20 days frominvasive outgrowth of tumors, primarily found in the mesen-teric lymph nodes. Immunization of mice with OVA which wasentrapped in liposome 1, liposome 2, or splenocytes indeedprovided protection against the subsequent EG7-OVA challenge and significantly prolonged their survival (P < 0.01)(Fig. 1). Mice vaccinated with free OVA or empty liposomeswere not primed for effective antitumor protection (P > 0.05).The best protection was observed in the mice that were primedwith OVA in splenocytes and boosted with liposomal OVA.Three of 5 mice survived >50 days before being sacrificed. Thelong term surviving mice did not possess any growing tumorswhen examined by necropsy. Furthermore, the immunizationprotocol protected the mice only from the challenge of antigen-expressing EG7-OVA cells; no protection was observed for EL4

6288




00

Days after tumor inoculation

Fig. 1. Immunoprotection of C57BL/6 mice challenged with EG7-OVA cells,by immunization with OVA entrapped in membranous vehicles. Mice were giveni.v. injections of OVA in liposome l (D), liposome 2 (O), or splenocytes (â€¢),freeOVA (A), or PBS (A) 7 days before the challenge with EG7-OVA cells. The valuesof mean survival time were 32.6, 30.6, 34.2, 19.8, and 18.2 days, respectively. Fivemice primed with OVA in splenocytes were boosted with OVA entrapped inliposome 1 (â€¢).Three of these mice survived >50 days before being sacrificed.

100

80-

60-1

40

20

10 20 30 40

Days after tumor innoculation

50 60

Fig. 2. Antigen-specific protection of the tumor-bearing mice by immunizationwith OVA encapsulated in membranous vehicles. Mice were immunized i.v. withOVA in splenocytes (squares), liposome 1 (circles), or PBS (triangles) and werechallenged with EG7-OVA cells (closed symbols) or EL4 cells (open symbols)1 days after the immunization. The prolongation of survival was observed only forthe mice inoculated with EG7-OVA thymoma. The values of mean survival timewere 46.2 days for the EG7-OVA-bearing mice immunized with OVA in splenocytes and 43.2 days for OVA in liposome 1, compared to 26.2 and 24.8 days,respectively, for the EL4-bearing mice. Untreated mice bearing either EG7-OVAor EL4 thymoma had a mean survival time of 19.6 and 19.8 days, respectively.

nization did not show much further improvement of their survival (P > 0.05).

Binding of Mouse Anti-OVA to EG7 Cells. Immunization ofC57BL/6 mice with liposomal OVA induced an OVA-specific

humoral response (Fig. 4). OVA entrapped in either liposome 1or liposome 2 stimulated higher levels of IgG production thandid free OVA. Since anti-OVA antibody might play a role in theprotective mechanism against the EG7-OVA cells, for instance,by complement-mediated cytolysis or antibody-dependent cellular cytotoxicity, we decided to investigate whether EG7-OVAcells express OVA epitopes on the cell surface for recognitionby anti-OVA sera. As shown in Table 1, the antibody to OVAcould not bind to EG7-OVA cells but did bind to EL4-OVAcells which had native OVA molecules artificially incorporatedon the cell membrane. Thus, the protection against tumor induction was unlikely to be due to the antibody response. Thelack of expression of native OVA on the surface of EG7-OVA

tumor-bearing mice (P > 0.05) (Fig. 2). Thus, the protectiveresponse was specifically against the OVA-expressing thymoma, indicating antigen specificity of the immunoprotection.

There was no improvement of the mean survival time observed in the mice treated with empty liposomes (P > 0.05)(Fig. 3). The minimal amount of liposomal OVA required toprime mice for a significantly prolonged survival was about 100Mg/mouse (P < 0.01). If the dose was <50 Mg/mouse, no significant protection against the challenge of EG7-OVA tumor cellswas observed (P > 0.05), compared to the empty liposometreatment. The result correlated well with the CTL activityelicited by the liposomal OVA; only marginal activity was obtained in mice treated with this low dose (data not shown).However, the mice receiving 250 tig liposomal OVA for immu-

6289

20-

0 10 20 30

Days after tumor innoculation

Fig. 3. Dose-dependent protective effect of OVA entrapped in liposomes. Micewere immunized i.v. with OVA in liposome 1 at a dose of 250 jig/mouse (A), 100Â¿ig/mouse(â€¢),or 50 Mg/mouse (â€¢)or with empty liposomes (D) or PBS (O). After7 days, the mice were challenged with EG7-OVA cells i.v. at a dose of 5 X IO6cells/mouse. The protection was observed in a dose-dependent manner. The values of mean survival time were 31.2, 28.4, 23.0, 19.8. and 18.6 days, respectively.

250n

200-

i 150-

~ 100-

20 30

Days After Immunization

40

Fig. 4. Humoral response elicited by immunization with liposomal OVA. Micewere immunized i.v. with OVA entrapped in liposome 1 (â€¢)or liposome 2 (â€¢)orsimply in PBS (D) and were boosted with the same forms of immunogens 14 daysafter the primary immunization. Concentrations of serum IgG were determinedby the ELISA method and are presented as ELISA units (absorbance x reciprocaldilutions). An enhanced IgG production was observed with both liposome formulations.




Table l Binding of mouse anti-OVA antibody lo EG7-OVA, EL4-OVA,and EL4 cells

For the antibody binding assay, cells were incubated with anti-OVA serumor PBS at 4'C for l h before addition of the I3ll-labeled secondary antibody.Percentage of antibody binding to cells is presented as mean Â±SD (n = 3).Anti-OVA did not bind to EG7-OVA cells significantly better than controls(P > 0.05). However, the anti-OVA serum bound to EL4-OVA cells much morethan to EG7-OVA cells and other controls (P < 0.01 ). For complement-mediatedcytolysis, cells were first incubated with anti-OVA or anti-Thy-1 at 4'C for 1 h,followed by a 1-h reaction with complement at 37Â°C.Anti-OVA again didnot sensitize EG7-OVA cells for complement-mediated lysis (P > 0.05). Aspositive controls, both EG7-OVA and EL4 cells were sensitized by anti-Thy-1 forcytolysis.

I31l-Antibody binding (%) 5lCr release (%)

CellsEL4

EL4OVAEG7-OVAAnli-OVA0.8

Â±0.412.5Â±2.12.2

Â±1.3PBS1.1

Â±0.81.9 Â±0.61.7 Â±0.9Anti-Thy-168.1

Â±0.8NDÂ°

61.8 Â±2.9Anti-OVA21.9

Â±3.5ND

18.3 Â±2.4'ND, not determined.

cells was further confirmed by the complement-mediated lysisassay. Both EG7-OVA and EL4 cells failed to fix complementin the presence of anti-OVA sera. However, they were lysed byaddition of anti-Thy-1 antibody and complement (Table 1).

Induction of Specific CTL Activity with OVA Encapsulatedin Different Membranous Vehicles and Its Correlation with theAntitumor Activity. The ability of various immunogens toprime for an OVA-specific CTL response and its correlation

with antitumor immunity is summarized in Table 2. As is apparent, OVA antigens encapsulated in the membranous vehicles, i.e., liposomes and splenocytes, induced an OVA-specificCTL response with varied efficiency. In agreement with previous observations (19), free OVA and empty liposomes failed toprime the mice for CTL production. Accordingly, mice immunized with free OVA and empty liposomes gained no protectionfrom the immunization (Fig. 1). The induced CTL activitykilled only EG7-OVA cells. Neither EL4 cells nor allogeneic

P815 cells osmotically loaded with OVA could be lysed, indicating that the CTL response was antigen specific and MHCclass I restricted. Liposome 1 had a pH-sensitive lipid compo

sition (22) and was designed to deliver protein antigens to thecytosol of antigen-presenting cells such that class I-restrictedpresentation was favored. The OVA-loaded pH-sensitive liposome induced a level of CTL response comparable to that induced by OVA-loaded splenocytes. Liposome 2, however, wasalso effective in inducing a CTL response, although the activityinduced appeared to be consistently lower than that induced byliposome 1. Booster protocols seemed to induce approximatelyequal levels of secondary CTL response but produced betterprotection, which may have resulted from the fact that an increased CTL precursor frequency was induced in the boostedmice (19). In general, CTL induction was important for mice toobtain protection. The frequency of CTL precursors but not thesecondary CTL activity was correlated with the prolonged survival of tumor-bearing mice.

If the injected dose was taken into consideration, OVA-

loaded splenocytes (25 tig/mouse) seemed to be more potent ineliciting an antitumor immune response than was OVA entrapped in liposomes (100 Mg/mouse). However, with the osmotic loading method, <1% of total OVA was taken up bysplenocytes, i.e., 5 mg of OVA were required to load 2.5 x IO7

splenocytes for one immunization of a single mouse. In contrast, the efficiency of loading OVA into liposomes was about25%. Only 250 ng of OVA were needed to load liposomes to

achieve the same efficiency for a single mouse. Thus, the liposome approach is more economical in the vaccination for anti-tumor activity.

DISCUSSION

Our previous studies have attested to the potential application of liposome formulations for antigen delivery to the class Ipathway of antigen-presenting cells (16, 17). This approachprovides a useful means to study the role of an antigen-specificcellular response in antitumor immunity. To simplify the complicated settings of real tumor immunology, we decided to testour liposome formulation initially in a model tumor system.OVA was chosen because it is a soluble protein antigen containing a known H-2Kb-restricted CTL epitope. This soluble

protein normally cannot be processed and presented with MHCclass I molecules. However, when OVA is introduced into thecytoplasm compartment by means of osmotic shock or pH-sensitive liposome-mediated delivery, it becomes available forclass I-restricted presentation (16, 18). Since OVA is easilydenatured and forms aggregates, we have optimized the preparation conditions and the improved method has achieved about25% entrapment efficiency, which allows more efficient sensi-tization of target cells (17). We then used the improved pH-sensitive liposomes to deliver OVA to the dendritic cells andsuccessfully induced a primary CTL response in vitro (23). Immunization of mice with liposomal OVA can also effectivelyinduce a secondary CTL activity in vivo ( 19). The induced CTLactivity is mostly from the CD8+ CTL population. These pre

vious results suggested that OVA is a good model antigen forthe study of the antitumor immune response. Since both EG7-OVA and EL4 cells express only class I molecules and not classII molecules, we were able to study specifically the CD8+ CTL-

mediated antitumor activity. Moreover, the EL4 cells do notsynthesize OVA, which allows us to examine whether the an-titumor response is antigen specific. Therefore, we believe thatthis model study is helpful for understanding the mechanism ofantitumor immunity and for designing tumor vaccines.

In the present study, we reported that liposomes can be usedas vaccine carriers to prime mice for antitumor immunity. Theprotection produced by immunization with liposomal antigenwas correlated with the induction of antigen-specific CTL response. In contrast, immunization with free OVA failed to

Table 2 CTL induction by and antitumor activity of different formsof immunogen

OVA antigen was either entrapped in liposomes with a freeze-thaw method orosmotically loaded in the cells or was simply in a free form. About 25 ng of OVAentrapped in the cells or 100 Â¿igof OVA in liposomes were injected per mouse toelicit the CTL response. Primed splenocytes were restimulated in vitro withEG7-OVA for 5 days and then used as effectors. Specific lysis at 50:1 effector totarget ratio is plotted. Spontaneous release was <15%. To determine the efficacyof the vaccination, mice that were challenged with EG7-OVA cells were checkedfor survival time (data taken from Fig. 1).

Specific lysis (%)

Antigen time(days)LIPOl(OVA)

LIP02(OVA)SPLEN(OVA)LIPOl(PBS)OVA (free)SPLEN + L1PO1"LIPO1(OVA)(3X)<-32.6

30.534.220.118.2ND*ND>50

days0/5

0/50/50/50/53/54/5EG7-OVA

cells EL4cells42.3

30.951.4

2.211.359.741.63.8

6.16.34.12.82.63.44.8

Â±3.31.3 Â±4.07.1 Â±2.13.1 Â±3.18.2 Â±4.5

10.5 Â±2.88.4 Â±2.2

" Mice were immunized with OVA entrapped in syngeneic splenocytes and

boosted once with LIPOl(OVA) 7 days after the injection.* ND, not determined.cMice were immunized with LIPOl(OVA) and boosted twice with the same

liposomal antigen at a 7-day interval.

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10.

15.

16.

induce CTL activity and no protection was observed. Thus, wesuggest that the liposomal OVA-induced CTL response in ourmodel system is the major mechanism for protection of EG7-OVA thymoma-bearing mice. Other forms of immunity, suchas NK cells, macrophages, antibody production, and secretionof cytokines, could also be elicited by liposomal antigen (24). Itis not likely that NK activity is the predominant mechanism inthis case because the class I-expressing EL4 or EG7-OVA cellsare mostly insensitive to NK cells (25). In fact, we found thatprimed splenocytes did not cytolyse either EL4 cells or NK- 8.sensitive YAC cells in vitro (data not shown), indicating that theNK activity was not induced. Macrophages in the liver and 9.spleen are responsible for the clearance of i.v. injected liposomes (26). Liposomes formulated with immunomodulatingagents could activate macrophages to a tumoricidal state (27).Since our empty liposomes did not improve the mouse survival(Fig. 3), these liposomes probably did not activate macrophages ' '

to kill tumor cells. Depletion of macrophages by liposomescontaining dichloromethylene diphosphonate (28) abolishes theCTL induction by liposomal OVA (29), suggesting that mac- 12

rophages are involved in the CTL induction but not in theactual killing of the tumor cells. Another important aspect of 13the antitumor mechanism is the antibody-mediated tumoricidalactivity, which was not detected in this model, although an 14antibody response to OVA antigen was induced (Fig. 4). OVAsynthesized in EG7-OVA cells was genetically engineered as asecretory protein (18); it presumably should not be present onthe cell surface in its native form. As demonstrated in the antibody binding and complement-mediated cytolysis assays, anti-OVA sera did not recognize EG7-OVA cells (Table 2). Theinduced humoral response was thus unlikely beneficial to themice bearing EG7-OVA thy moma. However, the enhanced an- 17tibody production may suggest that OVA-specific CD4+ T cellsare induced. The CD4+ T cells may play a role in helping CDS"1" 18

CTL induction (5) but they could not directly kill EG7-OVAtumors which do not express the class II molecules (18). Thuswe concluded that the CD8+ CTL activity primed by liposomal

OVA is most likely the primary mechanism to suppress theoutgrowth of EG7-OVA tumor in vivo and consequently prolonged the survival of the tumor-bearing mice.

In summary, we have demonstrated that our liposome approach is an effective means to induce a CTL response to amodel tumor-specific antigen. The antitumor immunity potentiated by the i.v. administered liposome formulation is protec- 22.live for the tumor-bearing mice. Since many malignant cells doexpress endogenous tumor-specific antigen but are poor anti- 23.gen-presenting cells by themselves, they are particularly ame

nable to an active immunotherapy protocol using defined antigen carriers such as liposomes. Of all vaccine carriers, weconsider our approach important because it is endowed withthe unique properties of well defined composition, simple prep- 2S

aration procedure, and a high degree of reproducibility. Withfurther optimization of the adjuvant activity of liposome for- 26.mutations, we anticipate that an effective vaccine for tumorprotection can be obtained.

27.

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1992;52:6287-6291. Cancer Res Fan Zhou, Barry T. Rouse and Leaf Huang Model Studywith a Soluble Protein Antigen Entrapped in Liposomes: A Prolonged Survival of Thymoma-bearing Mice after Vaccination

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