Liposomal Formulations of Synthetic MUC1 Peptides: Effects of

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Bioconjugate Chem. 1998, 9, 451−458

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ARTICLES Liposomal Formulations of Synthetic MUC1 Peptides: Effects of Encapsulation versus Surface Display of Peptides on Immune Responses Holly H. Guan,† Wladyslaw Budzynski,‡ R. Rao Koganty,‡ Mark J. Krantz,‡ Mark A. Reddish,‡ James A. Rogers,† B. Michael Longenecker,‡ and John Samuel*,† Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8, and Biomira Inc., Edmonton, Alberta, Canada. Received October 20, 1997; Revised Manuscript Received April 6, 1998

Synthetic human MUC1 peptides are important candidates for therapeutic cancer vaccines. To explore whether a human MUC1 peptide BP25 (STAPPAHGVTSAPDTRPAPGSTAPP) can be rendered immunogenic by incorporation in liposomes, the effects of physical association of the peptide with liposomes on immune responses were investigated. Lipid conjugated and nonconjugated MUC1 peptides were incorporated in liposomes with a composition of distearoylphosphatidylcholine/ cholesterol/dimyristoylphosphatidylglycerol (3:1:0.25, molar ratio) containing monophosphoryl lipid A (1% w/w of the total lipids). Liposomes were characterized for peptide retention by HPLC and for surface peptide display of MUC1 epitopes by flow cytometry. C57BL/6 mice were immunized with lipopeptide alone, peptide mixed with peptide-free liposomes, and peptide associated with liposomes in entrapped or surface-exposed forms. T cell proliferative responses, cytokine patterns, and antibody isotypes were studied. Results showed that immune responses were profoundly influenced by the liposome formulations. Physically associated, either encapsulated or surface-exposed, peptide liposomes elicited strong antigen-specific T cell responses, but not lipopeptide alone or peptide mixed with peptidefree liposomes. Analysis of the cytokines secreted by the proliferating T cells showed a high level of IFN-γ and undetectable levels of IL-4, indicating a T helper type 1 response. Thus, physical association of the peptide with liposomes was required for T cell proliferative responses, but the mode of association was not critical. On the other hand, the nature of the association significantly affected humoral immune responses. Only the surface-exposed peptide liposomes induced MUC1-specific antibodies. A domination of anti-MUC1 IgG2b over IgG1 (94 versus 6%) was observed. Our results support the hypothesis that different immune pathways are stimulated by different liposome formulations. This study demonstrated that a liposome delivery system could be tailored to induce either a preferential cellular or humoral immune response.

INTRODUCTION

MUC1 mucin is a high-molecular weight glycoprotein consisting of tandem repeats of the 20-amino acid unit PDTRPAPGSTAPPAHGVTSA (1). It is expressed by normal secretary epithelial cells and adenocarcinomas (2). MUC1 mucin produced by adenocarcinomas is underglycosylated in comparison to its normal counterpart. This results in exposure of certain core peptide epitopes of MUC1 to the immune system (3-6). Such epitopes may elicit anticancer immune responses in cancer patients, providing a rationale for the design of therapeutic MUC1 peptide cancer vaccines (7-9). Evidence for the protection against breast cancer through natural immunization by MUC1 epitopes during pregnancy suggests such vaccines may also have prophylactic applications (10-12). However, the problem of low immuno* Corresponding author. Telephone: (403) 492-7469. Fax: (403) 492-1217. E-mail: [email protected]. † University of Alberta. ‡ Biomira Inc.

genicity of synthetic MUC1 peptides must be overcome for the development of effective cancer vaccines. Synthetic MUC1 peptides coupled with a carrier protein have been shown to induce anticancer immune responses in mice and in cancer patients in phase I clinical studies (13; unpublished results). We have chosen liposomal delivery of MUC1 synthetic peptides as an alternate approach for enhancing immunogenicity. The immunoadjuvant properties of liposomes have long been recognized (14) and repeatedly demonstrated for humoral and cell-mediated immunity (15, 16). The nature of the physical association of protein antigens with liposomes can influence immune responses to a significant degree depending on the route of administration and antigen characteristics (17-20). In contrast to many studies on liposome-associated protein antigens, little work has been done with synthetic peptides, especially linear and hydrophilic peptides. Recently, a liposomal MUC1 peptide antigen delivery system containing monophosphoryl lipid A (MPLA) has been developed (21). It elicited strong antigen-specific T cell responses; however,

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no MUC1-specific antibodies were induced. Our working hypothesis is that the nature of physical association of MUC1 peptide with liposomes will influence the type of immune responses. The goal of this work was to evaluate the effects of the association of the MUC1 peptide with liposomes on antibody production and T cell responses. EXPERIMENTAL PROCEDURES

Materials. Mouse MUC1 peptide SP1-020 (DSTSSPVHSGTSSPATSAPEDSTS) and human MUC1 peptides BP16 (GVTSAPDTRPAPGSTA), BP24 (TAPPAHGVTSAPDTRPAPGSTAPP), and BP25 (STAPPAHGVTSAPDTRPAPGSTAPP) were constructed using the solidphase approach of Merrifield (22) on an automated peptide synthesizer (MilliGen/Biosearch model 9050). LCP25 (laurylcys-STAPPAHGVTSAPDTRPAPGSTAPP) was synthesized using laurylcysteine as the N-terminal amino acid. Laurylcysteine was presynthesized in solution phase by coupling lauric acid to the amino group of cysteine. Monoclonal antibodies (mAb) B248.7R2 specific for MUC1 peptide sequence APPAHGVTSA and B80.3 (specific for PSA, prostate-specific antigen) were produced and provided by Biomira Inc. (Edmonton, AB). Distearoylphosphatidylcholine (DSPC), cholesterol (CHOL), and dimyristoylphosphatidylglycerol (DMPG) were obtained from Princeton Lipids (Princeton, NJ). Peptide Liposome Formulations. Five formulations with equimolar peptide content (2 nmol per 50 µL of liposomes; 5 µg of BP25 or 5.6 µg of LCP25) were prepared for immune response studies. Multilamellar vesicles with a final lipid concentration of 30 mM, composed of DSPC/CHOL/DMPG (3:1:0.25, molar ratio) and MPLA (1% w/w of the total lipids) (Ribi Immunochem Inc., Hamilton, MT), were prepared by a modified freezeand-thaw (FAT) method (23, 24). Briefly, lipids were dissolved in chloroform and coated onto the walls of a 100 mL round-bottom flask by rotary evaporation, and the residual solvent was removed by overnight vacuum desiccation at 43 °C. The desiccated lipid film was dispersed in filter-sterilized PBS containing BP25 to form “liposomal BP25” at 65 °C followed by five cycles of FAT. Freezing was carried out in a dry ice/acetone bath for 5 min, and thawing occurred at room temperature for 50 min followed by incubation for 5 min at 65 °C. Free peptides in supernatants were removed after centrifugation at 150000g for 12 min (Beckman model L8-55 ultracentrifuge). Lipopeptide LCP25 was incorporated by mixing it with the lipids in chloroform prior to rotary evaporation, followed by hydration of the film with sterile PBS, forming liposomal LCP25. The entrapment of BP25 and LCP25 in liposomes was quantified by HPLC. Peptide-free liposomes (“empty”) containing 1% MPLA were similarly prepared, except that only PBS was used for hydration. The “BP25 mixed” group was formulated by adding a specific amount of BP25 to empty liposomes. A control formulation of lipopeptide LCP25 (“LCP25 alone”) was made by mixing it with 1% (w/v) MPLA in a PBS solution containing 0.1% (w/v) triethylamine. Determination of Peptide Leakage from Liposomes. Aliquots of 200 µL of liposome-encapsulated BP25 or LCP25 were incubated in a shaking water bath at 37 °C for 24 h. At various time intervals, liposome samples were centrifuged and analyzed by HPLC for MUC1 peptide in the supernatant and the pellet. Liposome Size Determination. Liposome size was determined by dynamic light scattering (Brookhaven Instruments, Holtsville, NY). Samples were diluted 300 times with buffer and measured at 25 °C.

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HPLC Analysis. Liposome pellets were dissolved in 25% absolute ethanol and then mixed with the internal standard phenol solution and PBS. A Waters 625-746 HPLC system with a C-18 reverse-phase radial-pak cartridge (8 mm × 100 mm) was used for HPLC analysis. Peptides were eluted using a gradient aqueous mobile phase consisting of eluent A [10% acetonitrile and 0.1% trifluoroacetic acid (TFA)] and eluent B (70% acetonitrile and 0.085% TFA). The flow rate was 1 mL/min. Peptides were detected by UV absorbance at 210 nm. Quantification of MUC1 peptides was done using a calibration curve based on peak areas (R2 > 0.9991). Flow Cytometric Analysis. To determine the surface exposure of MUC1 peptides, liposome samples (empty liposomes, BP25 mixed with empty liposomes, BP25 liposomes, and LCP25 liposomes) were incubated with B248.7R2 (MUC1-specific mAb) or B80.3 (negative control mAb) for 45 min at 4 °C, washed, and incubated with FITC-conjugated goat anti-mouse IgG1 (Southern Biotechnology Associates Inc., Birmingham, AL) at 4 °C for 30 min. After washing, liposomes were analyzed using a FACSort flow cytometer (Becton Dickinson, Mountain View, CA) under standard conditions. Immunization Procedures. Normal serum samples were collected from C57BL/6 mice (female, 7-8-weekold) 1 day before immunization by tail vein bleeding. Single Immunization. On day 0, five groups of mice (seven in each group) were immunized subcutaneously (sc) in the left hind footpad with 50 µL of the following five formulations: empty, BP25 mixed with empty liposomes, LCP25 alone, liposomal BP25, and liposomal LCP25. On day 10, blood was collected from each mouse by cardiac puncture and the draining popliteal lymphnodes were collected for the T cell proliferation assay. Two Immunizations. On day 0, another five groups of mice were immunized sc in the left thigh with the same formulations as above. On day 14, an identical second immunization was given intraperitoneally (ip). On day 26, blood and the draining inguinal lymph nodes were collected. T Cell Proliferation Assay and Cytokine Collection. Chemically inactivated syngenic spleen cells from naı¨ve mice were used as antigen-presenting cells (APCs). Mitomycin C (Sigma Chemical Co., Mississauga, ON) was added at a dose of 60 µg per 20 million spleen cells and the mixture incubated for 100 min at 37 °C in 5% CO2. T cells were isolated from the lymph node cells by the nylon wool purification method (25). T cells (2.5 × 105/well) and APCs (5 × 105/well) were cultured in a total volume of 300 µL in 96-well flat-bottom Costar microtiter plates (Fisher Scientific, Fair Lawn, NJ) in the presence of media alone (peptide-free background), SP1-020 (negative control), and BP24 or BP25 at 20 µg/well for a period of 3 days at 37 °C in 5% CO2. Each group was cultured in quadruplicate. On day 3, the cultures were pulsed with 1 µCi of [3H]thymidine (Amersham Canada Ltd., Oakville, ON) at 50 µL/well for 21 h. Incorporation of [3H]thymidine into the proliferating cells was measured after harvesting the cells on filters (Skatron model 11055 microcell harvester) and counting the filters (Beckman model LS 60001C liquid scintillation counter) in the presence of 4 mL of scintillation fluid (Cytoscint Research Production Division, Costa Mesa, CA). The stimulation index (SI) was defined as the ratio of the counts per minutes (cpm) of the peptide-stimulated cultures to the background cpm of peptide-free cultures. Separate parallel cultures under identical conditions were set up for collection of cytokines produced during T cell prolifera-

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Figure 1. FACS analysis of liposomal BP25 (A and B) and LCP25 (C and D) for the presence of MUC1 epitopes on the surface. Liposomes were incubated with negative control mAb B80.3 specific for PSA (A and C) or MUC1-specific mAb B248.7R2 specific for the amino acid sequence APPAHGVTSA (B and D), subsequently incubated with an appropriate FITC-labeled secondary antibody, and analyzed by FACS. The bars represent flourescence signal above background noise.

tion. Culture supernatant was collected from separate plates at 24, 48, and 72 h. Cytokine Analysis. Levels of INF-γ and IL-4 in the above culture supernatants were separately estimated at 24, 48, or 72 h by a sandwich type enzyme-linked immunosorbent assay (ELISA) (26, 27). The 96-well Nunc ELISA plates were coated with antibodies R46.A2 and 11B11 (Biomira Inc.) for IFN-γ and IL-4, respectively, by incubation at 37 °C for 30 min, followed by washing with TPBS (PBS with 0.05% Tween 20 at pH 7.4). Culture supernatants or recombinant reference standards (rIFN-γ, concentration range of 5000-156 pg/ mL; rIL-4, 1666-52 pg/mL; PharMingen, San Diego, CA) were added and the mixtures incubated at 37 °C for 45 min. After washing with TPBS, second antibodies (for IFN-γ, XMG1.2 biotinylated, Biomira Inc.; for IL-4, BVD6-24G2 biotinylated, PharMingen) were added and the mixtures incubated for 45 min at 37 °C. After washing, the wells were incubated with horseradish peroxidase (HRPO)-conjugated streptavidin (Jackson ImmunoResearch Lab Inc., Bar Harbor, ME) at 37 °C for 30 min and washed in TPBS. Finally, a HRPO substrate solution freshly prepared by mixing equal volumes of a TMB (3,3,5,5-tetramethylbenzidine) solution [0.4 g/L in an organic base, Kirkegaard and Perry Laboratories Inc. (KPL), Gaithersburg, MD] and a H2O2 solution (0.02% in citric acid buffer, KPL) were added to the wells (100 µL/well). The optical density (OD) at 650 nm was read immediately using a microplate reader (Molecular Devices, Menlo Park, CA) in a kinetic mode. A final OD reading was made at 450 nm after adding 100 µL of 1 M phosphoric acid to each well. Cytokine levels in the culture supernatants were determined on the basis of the standard curves generated for the reference standards. Antibody Analysis. Nunc maxisorb microplates were coated with 100 µL/well BP16-HSA (human serum albumin) and HSA (negative control) at a concentration of 5 µg/mL at 4 °C overnight. After washing with TPBS, the wells were blocked by incubation with bovine serum

albumin (1% in phosphate buffer, 200 µL/well, KPL) at 37 °C for 30 min and washing with TPBS. Then serial dilutions of test serum samples (pooled for each immunization group) were added to the wells (100 µL/well) and the mixtures incubated at room temperature for 1 h. A pooled normal serum sample (from unimmunized mice) was used as the negative control. Serum samples containing anti-MUC1 IgG and IgM from mice immunized with BP-16 conjugated to KLH (13) were used as positive controls (serial dilutions for IgM, 1:80 to 1:5120; for IgG, 1:2500 to 1:160000). The plates were then washed with TPBS. Goat anti-mouse IgG and IgM antibodies labeled with HRPO (KPL) at a 1:2000 dilution in PBS or subtype-specific goat anti-mouse IgG1, IgG2a, IgG2b, and IgG3 antibodies labeled with HRPO (Southern Biotechnology Associates Inc.) at a 1:4000 dilution in PBS were added to each well (100 µL/well). After incubation at room temperature for 1 h and subsequent washing with TPBS, a HRPO substrate solution freshly prepared by mixing equal volumes of an ABTS [2,2′azinobis(3-ethylbenzthiazoline sulfonate)] solution (0.6 g/L in glycine buffer, KPL) and a H2O2 solution (0.02% in citric acid buffer, KPL) were added to the wells (100 µL/well). The wells were incubated at room temperature for 15 min, and the OD was read at 405 nm using a microplate reader (Molecular Devices). RESULTS

Physical Characterization of Liposomes Containing Peptide versus Lipopeptide Antigen. FACS analysis of liposomal LCP25 with a mAb B248.7R2 (specific for the amino acid sequence APPAHGVTSA of human MUC1 mucin) clearly showed that the MUC1 peptide epitopes were present on the surfaces of liposomes, whereas no MUC1 epitopes were detected on liposome surfaces in the rest of the formulations (Figure 1). An irrelevant mAb B80.3 (specific for PSA) did not bind with LCP25 liposomes, establishing the antigen specificity of the FACS analysis. BP25 and LCP25

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Figure 2. Retention of BP25 and LCP25 in liposomes with a lipid composition of 3:1:0.25 DSPC/CHOL/DMPG at 37 °C in PBS over the course of 24 h. Error bars indicate the standard deviation (SD) (n ) 3).

liposomes leaked less than 10% of the entrapped peptides over the course of 24 h at 37 °C (Figure 2). The mean diameters of BP25 and LCP25 liposomes were 0.8 ( 0.1 and 0.9 ( 0.1 µm, respectively. Physical Association of the MUC1 Peptide with Liposomes Was Required for T Cell Responses. Antigen-specific T cell priming induced by liposomal formulations was studied by T cell proliferation assays. T cell responses (SI) were significantly stronger (P < 0.01) in groups of mice immunized with the peptide-associated liposomes (encapsulated or surface-exposed) than in the rest of the formulations (Figure 3). There were no significant differences (P > 0.01) between the encapsulated and surface-exposed liposomes or between the two different immunization procedures (Figure 3). Antigen specificity was established by the observation that T cells responded to both recall human MUC1 peptides BP24 and BP25 but not to the irrelevant mouse MUC1 peptide SP1-020. Cytokine Analysis Indicated a T Helper Type 1 (Th1) Response. T cell responses were further characterized by analyzing the cytokine secretion profiles of the proliferating T cells. No evidence of IL-4 was present in the T cell cultures following one or two immunizations, at 24, 48, or 72 h (all below the detectable level of 52 pg/mL; data not shown). However, there were significant levels of IFN-γ in the cell cultures that correlated with T cell responses (Figures 3 and 4). The amount of IFN-γ was found to be highest at 72 h. These results are indicative of Th1 responses (28). Surface-Exposed but Not Encapsulated MUC1 Peptide Liposomes Induced Antibody Production. MUC1-specific humoral immune responses were evaluated by ELISA using BP16-HSA as the solid phase. BP16 contains the immunodominant B cell epitopes of MUC1 tandem repeat sequences in mice (13). The positive control serum for IgG and IgM was from mice immunized with BP16-KLH (keyhole limpet hemocyanin). There were no detectable levels of IgG or IgM after one immunization (Figure 5). However, high titers of IgG and IgM were detected in the sera pooled from the group of mice immunized twice with liposomal LCP25, but not with other formulations (Figure 6). This serum was further analyzed for IgG subclasses IgG1, IgG2a, IgG2b, and IgG3. Results showed a domination of IgG2b over IgG1 (94 versus 6%) (Figure 7). There were no detectable levels of IgG2a or IgG3. DISCUSSION

Our results showed that physical association of the MUC1 peptide with liposomes containing MPLA was

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necessary for induction of immune responses. Both encapsulated and surface-displayed formulations of peptides induced T cell responses, whereas an aqueous solution of lipopeptide containing MPLA or free peptide mixed with liposomes failed to induce antibodies or T cell responses. For most protein antigens, potent humoral responses may be induced by a variety of liposomal formulations, where protein antigens are entrapped, surface-linked, or even mixed with liposomes (29-31). Protein antigens containing hydrophobic domains may physically associate with lipid bilayers even by simple mixing with liposomes. Hydrophilic peptides such as MUC1 peptides used in our study are unlikely to associate with lipid bilayer. This is confirmed by the lack of MUC1-specific antibody binding with BP25 encapsulated liposomes. Since short peptides are quickly degraded by peptidases and rapidly cleared, they are unlikely to induce immune responses unless they are delivered in physical association with liposomes. The ability of the liposomes to retain more than 90% of the MUC1 peptides at 37 °C suggests that a major fraction of the peptides will be delivered to the immune system in an associated rather than released form. MUC1 peptide delivery by both encapsulated or surfaceexposed liposome formulations was equally effective in priming T cell responses in vivo. Induction of T cell responses requires antigen uptake by professional APCs and presentation of the antigenic peptide in association with MHC molecules: MHC class II presentation for CD4+ T helper (Th) cells and MHC class I presentation for CD8+ cytotoxic T lymphocytes (CTLs) (32). Liposomes are efficient vehicles for antigen delivery to professional APCs (33). Phospholipids have been shown to facilitate antigen presentation by enhancing peptide binding to MHC class II molecules possibly by inducing conformational changes in MHC class II molecules (34). Liposomes are also efficient vehicles for induction of CTLs (35, 36). The strong antigen-specific T cell proliferation in response to MUC1 peptides ex vivo is an indication of successful in vivo priming of Th cells by immunization with the liposomal formulations. It is possible that CD8+ CTLs also might have contributed to the proliferative responses ex vivo. However, further studies on the CD4/ CD8 profile of the activated T cells and MHC-restricted lysis of MUC1+ target cells are required to confirm in vivo priming of CTLs by immunization with the liposomal MUC1 peptide formulations. The cytokine secretion pattern (IFN-γ but not IL-4) of the MUC1-specific T cells indicates that the immune responses induced by both liposomal formulations were of the T helper 1 (Th1) type (28). It has been previously proposed that liposome surface-exposed antigen preferentially follows a Th1 and encapsulated antigen follows a Th2 activation pathway on the basis of the immunoglobulin (Ig) isotype profiles (37). Another report has suggested that both encapsulated and surface-linked formulations induce a Th1 type immunity (31). The exact mechanisms by which our formulations bias the immune responses toward a Th1 type have not been examined. We hypothesize that antigen delivery by these formulations results in induction of IL-12, a key cytokine initiator of the Th1 pathway of immune response. Phagocytosis of liposomes by professional APCs may induce secretion of IL-12. Uptake of particulate materials has been reported to induce IL-12 secretion by macrophages and dendritic cells (38). Further, MPLA adjuvant may favor Th1 responses by induction of IL-12 secretion by APCs (39). The mode of physical association of MUC1 peptide with

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Figure 3. T cell responses induced by MUC1 peptide formulations after one or two immunizations (peptide dose, 2 nmol/mouse). T cells purified from lymph nodes were cultured along with antigen-presenting cells alone or in the presence of human MUC1 peptides (BP24 or BP25) or an irrelevant mouse MUC1 peptide (SP1-020) as shown. The stimulation index (SI) was defined as the ratio of counts per minute (cpm) of the peptide-stimulated cultures to the background cpm of peptide-free cultures. Error bars indicate the SD (n ) 4).

Figure 4. Levels of IFN-γ in the 72 h T cell cultures from groups of mice immunized with the MUC1 peptide formulations. T cells purified from lymph nodes were cultured along with antigen-presenting cells alone or in the presence of human MUC1 peptides (BP24 or BP25) or an irrelevant mouse MUC1 peptide (SP1-020) as shown. Error bars indicate the SD (n ) 4).

Figure 5. Anti-MUC1 IgG and IgM analysis from sera pooled from each group of mice immunized once (sc) with MUC1 peptide formulations. The polyclonal antiserum from BP16-KLH-immunized mice served as the positive control. The optical density (OD) was determined at a wavelength of 405 nm. Error bars indicate the SD (n ) 3).

liposomes was found to be a key element for the humoral immune response induction. Only one formulation, LCP25 (N-terminally acylated BP25) liposomes with

surface-exposed MUC1 epitopes, elicited antibody production. No detectable antibody responses were induced by the BP25 encapsulated formulation, even though the

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Figure 6. Anti-MUC1 IgG and IgM analyses from sera pooled from each group of mice immunized twice (sc and ip, 14 days apart) with MUC1 peptide formulations. The polyclonal antiserum from BP16-KLH-immunized mice served as the positive control. The optical density (OD) was determined at a wavelength of 405 nm. Error bars indicate the SD (n ) 3).

Figure 7. Anti-MUC1 IgG isotype analyses from sera of the group of mice immunized twice with liposomal LCP25. The optical density (OD) was determined at a wavelength of 405 nm. Error bars indicate the SD (n ) 3).

immunization routes (sc followed by an ip booster) were chosen to favor antibody production. These results are consistent with our previous studies where a MUC1 24mer peptide (BP24) encapsulated within liposomes also failed to induce detectable antibodies at a comparable dose (5 µg/mice) after three sc immunizations (21). White et al. (35) reported that a gp120 V3 loop peptide (P18) encapsulated in liposomes induced CTL responses, but not detectable antibody responses (35). Induction of antibody responses against V3 loop epitopes required presentation of the epitopes on the liposome surface. Antibody responses are initiated by B cell recognition of antigens through Ig receptors on their surface (40, 41). The lack of antibody responses against liposome-encapsulated MUC1 peptide may be due to the lack of antigen accessibility to the Ig molecules on B cells. Both encapsulated and surface-exposed peptide liposomes would be expected to be phagocytosed and processed by professional APCs such as dendritic cells and macrophages; however, only the liposomes with surface-exposed peptide would interact with B cells. Dal Monte and Szoka demonstrated that soluble and liposome surface-exposed but not encapsulated pigeon cytochrome c (PCC) antigen was processed and presented by B cells in vitro (42), whereas the encapsulated PCC was phagocytosed and

presented by macrophages (43). Thus, the lack of antibody responses against the liposome-encapsulated MUC1 peptide may be due to the lack of antigen accessibility to the Ig molecules on B cells. Another important factor influencing antibody responses is the epitope repetitiveness. Repetitiveness of viral epitopes is a key factor responsible for efficient cross linking of surface Ig receptors leading to strong B cell responses, including isotype switching (44). The LCP25 liposomes would be expected to present multiple surface-exposed MUC1 epitopes to B cells, leading to efficient B cell activation through crosslinking of surface Igs. B cell recognition of MUC1 epitopes in BP25 liposomes would be dependent on the slow release of the encapsulated peptide, which would be detected only as monomers. Another factor influencing the differential B cell responses might be the higher in vivo stability of the lipopeptide in comparison to the unmodified peptide (BP25) (45, 46). Immunoglobulins induced by liposomal LCP25 included IgM and IgG, with a domination of IgG2b over IgG1 (94 versus 6%) and no detectable levels of IgG2a or IgG3. The induction of Ig isotypes is influenced by many factors, including antigen location, adjuvants, mice strain, and route of administration (18, 19, 47). Immunomodulatory properties of adjuvants can affect Ig isotype distribution. Enhanced expression of IgG2b over IgG1 was observed by using lipid-soluble MDP (muramyl dipeptide) liposome-encapsulated protein antigens, but not with water-soluble MDP liposomes (48). Using liposomal surface-conjugated peptides, Friede et al. also observed a predominant production of IgG2b over IgG1 (49). Dominance of IgG2a and/or IgG2b over IgG1 is associated with Th1 type immune responses and is augmented by IFN-γ (50, 51). The ratio of IgG2a:IgG2b titers is influenced by the mouse strain, with a predominance of IgG2a in A/J mice and a predominance of IgG2b in C57BL/6 mice (47, 52). The gene corresponding to IgG2a in C57BL/6 mice (IgG2c) is significantly different from that in Balb/c, and the commercial reagents for murine IgG2a might not cross-react substantially with this isotype in C57BL/6 strains (53, 54). Therefore, IgG2b responses are a better indicator of Th1 responses

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in C57BL/6 mice. Thus, the isotype distribution of humoral immune responses also confirmed that Th1associated immune responses were induced by liposomal LCP25. In summary, immune responses were profoundly influenced by characteristics of the liposome formulations. This study demonstrated that liposome delivery systems could be tailored to induce preferential cellular or humoral immune response. Our results suggest that encapsulation of peptide antigens in liposomes offers an approach to selective induction of T cell responses without activation of the humoral responses. Alternatively, surface display of peptides on liposomes would allow activation of humoral responses without compromising T cell responses. Such selective immune manipulations are useful in defining the protective immune responses against infections and cancer and are of special significance to therapeutic cancer vaccines. The results of this study are also relevant to vaccine design in general. ACKNOWLEDGMENT

We thank Ms. Deborah Sosnowski, Ms. Ann Burrell, Ms. Brenda Christian, and Mr. Aldo Ritacco for their excellent technical assistance. Financial support for Ms. Holly Guan was provided by a Province of Alberta Postgraduate Fellowship. We thank the Medical Research Council of Canada for financial support of this work through a research grant to Dr. John Samuel (MT13261). LITERATURE CITED (1) Gendler, S., Taylor-Papadimitriou, J., Duhig, T., Rothbard, J., and Burchell, J. (1988) A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats. J. Biol. Chem. 263, 1282012823. (2) Zotter, S., Hageman, P. C., Lossnitzer, A., Mooi, W. J., and Hiljers, J. (1988) Tissue and tumor distribution of human polymorphic epithelial mucin. Cancer Rev. 11, 55-58. (3) Burchell, J., Gendler, S., Taylor-Papadimitriou, J., Girling, A., Lewis, A., Millis, R., and Lamport, D. (1987) Development and characterization of breast cancer reactive monoclonal antibodies directed to the core protein of the human milk mucin. Cancer Res. 47, 5476-5482. (4) Hull, S. R., Bright, A., Carraway, K. L., Abe, M., Hayes, D. F., and Kufe, D. W. (1989) Oligosaccharide differences in the DF3 sialomucin antigen from normal human milk and the BT-20 human breast carcinoma cell line. Cancer Commun. 1, 261-267. (5) Finn, O. J. (1992) Antigen-specific, MHC-unrestricted T cells. Biotherapy 4, 239-249. (6) Jerome, K. R., Domenech, N., and Finn, O. J. (1993) Tumorspecific cytotoxic T cell clones from patients with breast and pancreatic adenocarcinoma recognize EBV-immortalized B cells transfected with polymorphic epithelial mucin complementary DNA. J. Immunol. 151, 1654-1662. (7) Longenecker, B. M., and MacLean, G. D. (1993) Prospects for mucin epitopes in cancer vaccines. Immunologist 1, 8993. (8) Samuel, J., and Longenecker, B. M. (1995) Development of Active Specific Immunotherapeutic Agents Based on Cancerassociated Mucins. In Vaccine design-the subunit and adjuvant approach (M. F. Powell and M. J. Newman, Eds.) pp 875-890, Plenum Press, New York. (9) Finn, O. J., Jerome, K. R., Henderson, R. A., Pecher, G., Domenech, N., Magarian-Blander, J., and Barratt-Boyes, S. M. (1995) MUC-1 epithelial tumor mucin-based immunity and cancer vaccines. Immunol. Rev. 145, 61-89. (10) Agrawal, B., Reddish, M. A., Krantz, M. J., and Longenecker, B. M. (1995) Does pregnancy immunize against breast cancer? Cancer Res. 55, 2257-2261.

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