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The Journal of Immunology, 2008, 180, 3719 -3728
Copyright © 2008 by The American Association of Immunologists, Inc.
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The Use of Filamentous Bacteriophage fd to Deliver MAGE-A10 or MAGE-A3 HLA-A2-Restricted Peptides and to Induce Strong Antitumor CTL Responses1

Rossella Sartorius*, Paola Pisu{dagger},§, Luciana D’Apice*, Luciano Pizzella*, Chiara Romano{dagger},§, Giancarlo Cortese{dagger},{ddagger}, Angela Giorgini{dagger}, Angela Santoni{dagger},§, Francesca Velotti2,{dagger},¶ and Piergiuseppe De Berardinis2,3,*

* Institute of Protein Biochemistry, Consiglio Nazionale delle Ricerche, Naples; {dagger} Centro Ricerca Sperimentale and {ddagger} Stabilimento Allevatore Fornitore Utilizzatore Department, Regina Elena Cancer Institute, Rome; § Department of Experimental Medicine and Pathology, "La Sapienza" University, Rome; and Department of Ecology and Economic Sustainable Development, Tuscia University, Largo dell’Università, Viterbo, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Delivery of tumor-associated Ag-derived peptides in a high immunogenic form represents one of the key issues for effective peptide-based cancer vaccine development. We report herein the ability of nonpathogenic filamentous bacteriophage fd virions to deliver HLA-A2-restricted MAGE-A10254–262- or MAGE-A3271–279-derived peptides and to elicit potent specific CTL responses in vitro and in vivo. Interestingly, human anti-MAGE-A3271–279-specific CTLs were able to kill human MAGE-A3+ tumor cells, even if these cells naturally express a low amount of MAGE-A3271–279 peptide-HLA epitope surface complexes and are usually not recognized by CTLs generated by conventional stimulation procedures. MAGE-A3271–279-specific/CD8+ CTL clones were isolated from in vitro cultures, and their high avidity for Ag recognition was assessed. Moreover, in vivo tumor protection assay showed that vaccination of humanized HHD (HLA-A2.1+/H2-Db+) transgenic mice with phage particles expressing MAGE-A3271–279-derived peptides hampered tumor growth. Overall, these data indicate that engineered filamentous bacteriophage virions increase substantially the immunogenicity of delivered tumor-associated Ag-derived peptides, thus representing a novel powerful system for the development of effective peptide-based cancer vaccines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In the past decade, the identification and molecular characterization of many human tumor-associated Ags (TAAs)4 recognized by CTLs has led to the development of new immunotherapeutic strategies of vaccination aimed at producing antitumoral CTL responses in cancer patients (1, 2, 3, 4). Among TAAs, cancer/testis Ags, being expressed in many tumors of various histological types but not in normal tissues (with the exception of testis and placenta), are strictly tumor-specific and therefore ideal candidates for cancer vaccines (1, 4). Cancer/testis Ags include the MAGE family, constituted of several related Ags divided into three clusters, namely MAGE-A, MAGE-B, and MAGE-C, and they are expressed in a large variety of primary as well as metastatic tumors (2, 5, 6). In particular, MAGE-A3 and MAGE-A10 Ags have elicited considerable interest because they are expressed with high frequency in melanomas (~70% for MAGE-A3 and 50% for MAGE-A10) and in bladder, lung, esophagus, and head and neck carcinomas (~50% for MAGE-A3 and 35% for MAGE-A10) (2, 5, 6, 7). A variety of peptide epitopes present in the amino acid sequences of MAGE-A3 and MAGE-A10 Ags have been characterized (8, 9), and special interest has been bestowed upon HLA-A2 as a restriction element because of its frequency of occurrence in various ethnic groups (10). The nonapeptide-encompassing residues 271–279 and 254–262 from MAGE-A3 (MAGE-A3271–279) and MAGE-A10 (MAGE-A10254–262), respectively, are recognized by CTLs restricted by HLA-A2 (11, 12). Studies on T cell responses to MAGE-A3271–279 and MAGE-A10254–262 peptides, even in association with cytokines or presented by dendritic cells as APCs, have shown that specific CTL responses required repeated stimulations in vitro (11, 12, 13, 14, 15, 16) and that repeated immunizations rarely generated CTL responses in vivo (17, 18, 19, 20, 21). Moreover, when the generation of peptide-specific CTLs could be achieved, CTLs might fail to recognize the peptide epitope on the neoplastic cell. This was the case of MAGE-A3271–279 peptide-specific CTLs, which readily lysed HLA-A2+/MAGE-A3271–279 peptide-loaded target cells, whereas they did not recognize HLA-A2+/naturally expressing MAGE-A3 tumor cells (13, 22). The lack of efficient tumor cell recognition by MAGE-A3271–279 peptide-specific CTLs was due to the low abundance of peptide-HLA epitope complexes on the tumor cell surface, because of an impaired peptide processing in the neoplastic cell (22, 23). Taken together, these observations raise concerns on the immunogenicity of these MAGE peptide epitopes and, hence, on their usefulness as vaccines. Thus, the possibility to deliver TAA peptides in a high immunogenic form, capable of eliciting not only specific but also potent CTL responses able to recognize low amounts of Ag on the tumor cell, represents one of the key issues for the development of more effective peptide-based cancer vaccines.

Herein, we propose a novel Ag delivery system based on benign filamentous bacteriophage fd virions. We have described the ability of fd virions, engineered to display multiple copies of foreign peptide-surface epitopes, to evoke both B cell- and T cell-mediated immune responses (24). Antigenic peptides can be expressed on the capsid of the filamentous bacteriophage fd, in the exposed N-terminal region of the 2700 copies of the major coat protein pVIII, and elicit the production of high titers of Abs and/or the generation of Th cell responses (24, 25, 26, 27, 28). Moreover, we have demonstrated that double-hybrid filamentous bacteriophages, coexpressing Th cell and CTL epitopes from HIV-1-RT on the same capsid, elicited a Th-dependent anti-HIV-1 CTL response (29).

In this study we constructed double-hybrid filamentous bacteriophage fd coexpressing the promiscuous HLA-DR-restricted Th cell peptide epitope p23 from the HIV-1-RT- (29, 30) and the HLA-A2-restricted CTL peptide MAGE-A10254–262 (12) or MAGE-A3271–279 (11). We demonstrate that in vitro stimulation of human PBMCs and immunization of HHD (HLA-A2.1+/H2-Db+) transgenic mice with these peptides delivered via filamentous bacteriophage fd virions elicited specific and potent CTL responses. We also observed induction of tumor protection in HHD mice vaccinated with phage particles expressing the MAGE-A3 antigenic determinant. Overall, our data indicate that engineered filamentous bacteriophage virions represent a novel versatile and powerful peptide delivery system for the development of effective peptide-based cancer vaccines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Construction and purification of double-hybrid bacteriophage virions

Double-display fd23/Mg10 and fd23/Mg3 bacteriophage virions coexpressing the Th cell peptide (KDSWTVNDIQKLVGK) p23 from the HIV-1-RT, which is promiscuously recognized by several DR alleles (29, 30), and the HLA-A2-restricted CTL nonapeptide-comprising residues 254–262 (GLYDGMEHL) Mg10 or 271–279 (FLWGPRALV) Mg3 from MAGE-A10 (12) and MAGE-A3 (11) TAAs, respectively, were constructed as described elsewhere (29, 31). Oligonucleotide sequences encoding MAGE-A10254–262 or MAGE-A3271–279 peptides flanked by the sequences for the +2–+3 and +4–+10 residues of the pVIII protein and by the 5' protruding ends of SacII-StyI restriction sites were designed. Each pair of sequence was annealed and ligated into bacteriophage fdAMPLAY88 DNA, previously digested with SacII and StyI restriction enzymes. The DNA were transformed into Escherichia coli TG1 recO cells, and their identities were confirmed by DNA sequencing. Thereafter, the single-display fdMg10 or fdMg3 bacteriophages were purified and used to infect E. coli TG1 recO cells previously transformed with the plasmid pTfd8p-66 (31), which encoded p23 incorporating pVIII proteins. The double-hybrid virions were harvested, purified on cesium chloride gradient, and analyzed by SDS-PAGE. The numbers of copies of pVIII displaying p23, MAGE-A10254–262, or MAGE- A3271–279 peptides were calculated based on the relative yields of the various N-terminal sequences obtained by direct sequence analysis of the purified virions, as previously described (32).

Cell lines, tumors, and synthetic peptides

The human 1512 short-term cultured urinary bladder transitional-cell carcinoma (TCC) cell line was generated by direct culturing of a resected bladder TCC tissue (33). The human T24, J82, and SD48 long-term established bladder TCC cell lines were obtained from originators’ laboratories or from American Type Culture Collection. The human 10538, M14, and SK23-MEL melanoma cell lines were gifts from Dr. P. Nisticò and Dr. G. Zupi (Regina Elena Cancer Institute, Rome, Italy) and Dr. C. Traversari (San Raffaele Institute, Milan, Italy), respectively. All of these tumor cell lines were characterized for HLA-A2, MAGE-A10, and MAGE-A3 Ag expression (Table I). The HLA-A2 haplotype was assessed at both DNA level using the Micro SSP HLA Class I kit (One Lambda, Hoffman-La Roche) and protein level by immunofluorescence and flow cytometry using the anti-HLA-A2.1 BB7.2 mAb (BD Biosciences). MAGE-A10 and MAGE-A3 expression were analyzed at the mRNA level by RT-PCR, using the following sense and antisense primers: for MAGE-A10: 5'-CAC AGA GCA GCA CTG AAG GAG-3' and 5'-CTG GGT AAA GAC TCA CTG TCT GG-3'; for MAGE-A3: 5'-TGG AGG ACC AGA GGC CCC C-3' and 5'-GGA CGA TTA TCA GGA GGC CTG C-3' (5). For some relevant tumor cell lines, MAGE-A3 transcript expression levels were analyzed by real-time PCR analysis using the following sense and antisense primers: for MAGE-A3: 5'-GGC TCG GTG AGG AGG CAAG-3' and 5'-GAT GAC TCT GGT CAG GGC AA-3'; and for GAPDH: 5'-ACA TGT TCC AAT ATG ATT CCA-3' and 5'-TGG ACT CCA CGA CGT ACT CAG-3' (34). PCR amplifications were done with iQ SYBR Green Supermix (Bio-Rad), and quantitative measurements of specific transcripts were acquired using ICycler iQ real-time detection system (Bio-Rad). To verify that a single product was amplified, a melting curve was generated at the end of every run by slowly increasing the temperature from 72 to 95°C. The relative expression levels were calculated by the comparative cycle threshold (CT) method and normalized by GAPDH expression. The N-fold differential expression of MAGE-A3 was expressed as 2–{Delta}{Delta}CT, where CT was defined as the cycle number at which the amount of amplified target reached a fixed threshold, {Delta}CT was the difference in the CT values of MAGE-A3 and GAPDH for each cell line, and {Delta}{Delta}CT represented the difference between {Delta}CT of each sample and the {Delta}CT of SK23-MEL, because this latter cell line expressed the lowest value of MAGE-A3 transcripts. For proteasome inhibition experiments, cell lines were treated with 10 µM of lactacystin (Calbiochem) for 17 h at 37°C. The human CEMx721.174.T2 (T2) (Tap–/–, HLA-A2.1+, HLA class II–) (35) and the murine RMA-S-HHD (Tap–/–, transfected with the HLA-A2.1/H2-Db monochain) (36) cell lines were used for cell peptide-loading experiments. The murine EL4S3-Rob-HHD (β2m-/HLA-A2.1/H2-Db monochain) (36) cell line was a kind gift of Dr. F. A. Lemonnier (Pasteur Institute, Paris, France). All cell lines were maintained in RPMI 1640 medium (BioWhittaker Europe) supplemented with 10% FCS, except for J82 cells that were grown in DMEM (BioWhittaker Europe). The RMA-S-HHD and the EL4S3-Rob-HHD cell lines were maintained in RPMI 1640 medium supplemented with 1 mg/ml G418 (Calbiochem). For the construction of the EL4-HHD/MAGE-A3 cell line, cDNA encoding for the full-length MAGE-A3 was cloned into the pcDNA 3.1/Hygro+ vector (Invitrogen Life Technologies) between the XbaI-NotI restriction sites. EL4-HHD cells (5 x 106) were transfected by electroporation with 20 µg of pcDNA 3.1/Hygro+-MAGE-A3 plasmid at 250 mV and 950 µF. After 48 h, cells were cloned by limiting dilution in RPMI 1640 medium containing 200 µg/ml hygromicin B (Calbiochem). Hygromicin B/G418-resistant clones, expressing the MAGE-A3 protein, were identified by RT-PCR using the MAGE-A3 primers described above. Tumors derived from EL4-HHD/MAGE-A3 cell line inoculations were analyzed for HLA-A2 and MAGE-A3 expressions by Western blot analysis using the anti-HLA-A2 HCA2 mAb (37) (kindly provided by Dr. H. Ploegh, Whitehead Institute for Biomedical Research, Cambridge, MA) and by RT-PCR using the MAGE-A3 primers described above, respectively. The MAGE-A10254–262 and MAGE-A3271–279 synthetic peptides were purchased from the University of Lausanne (Lausanne, Switzerland).


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  		Table I. Expression of HLA-A2, MAGE-A10, and MAGE-A3 Ags by tumor cell lines

 
Human PBMCs and generation of CTLs in vitro

PBMCs were isolated from the venous blood of three different HLA-A2+ healthy donors (LDA, ACN, and ACA) using Ficoll-Paque PLUS (Amersham Biosciences) gradient. PBMCs were allowed to adhere to 2% gelatin-coated plates at a density of 106 cells/cm2. Adherent PBMCs were separated from nonadherent PBMCs and used as APCs. APCs (300,000) were pulsed for 3 h at 37°C with 50 µg/ml double-display fd23/Mg10 or fd23/Mg3 bacteriophage virions or, as a control, with 50 µg/ml wild-type bacteriophage fd virions in the presence of 1.2 µg/ml p23 and 0.35 µg/ml MAGE-A10254–262 or MAGE-A3271–279 synthetic peptides. The concentration of 50 µg/ml bacteriophage fd virions has been established to be an optimal dose, by culturing PBMCs (from donor LDA) with APCs pulsed with increasing concentrations (from 1 to 100 µg/ml) of fd23/Mg3 double-display bacteriophage virions (data not shown). Pulsed APCs were {gamma}-irradiated (4000 rad), washed, and cocultured with 106 nonadherent PBMCs (as a source of CTLs) in RPMI 1640 supplemented with 10% autologous human serum. After 24 h, 10 U/ml human rIL-2 (Boehringer Mannheim) were added to the cultures. After 2 wk, growing cell lines were restimulated with the above-mentioned Ags and autologous {gamma}-irradiated PBMCs as APCs. Nine days after the second stimulation, effector cells were harvested and assayed for their proliferation, cytotoxic activity, and pentamer staining.

Isolation of CD8+ T cell clones

CD8+ T cells were sorted from the MAGE-A3-specific (donor) LDA T cell line using anti-CD8-labeled magnetic beads (Miltenyi Biotec), and they were cloned by limiting dilution at 0.5 cells/well in the presence of irradiated autologous PBMCs, 0.5 µg/ml PHA (Boehringer Mannheim), rIL-2 (20 U/ml), and IL-7 (10 ng/ml) (Peprotech). Growing CD8+ clones were expanded in 24-well plates and periodically restimulated using the same protocol. Ten days after restimulation, CD8+ T cell clones were harvested and assayed for cytotoxic activity and pentamer staining.

Mice and generation of CTLs in vivo

HHD transgenic mice (36) were kindly provided by Dr. F. A. Lemonnier. They express a chimeric HLA-A2.1/H2-Db MHC class I monochain on a C57BL/6 genetic background. The mice were housed in a temperature-controlled, light-cycled room. All the in vivo experiments were approved by the Institutional Review Committee. HHD mice were immunized at days 0 and 14 by injecting s.c. at the base of the tail 140 µg of double-display fd23/Mg10 or fd23/Mg3 bacteriophage virions emulsified (v/v) in Incomplete Freund’s adjuvant or, as a control, with 140 µg of wild-type bacteriophage fd virions in the presence of 3.4 µg of p23 and 0.98 µg of MAGE-A10254–262 or MAGE-A3271–279 synthetic peptides coemulsified in Incomplete Freund’s adjuvant. After 7 days, mice were sacrificed and isolated splenocytes (5 x 106) were cocultured with Ag-pulsed {gamma}-irradiated (10,000 rad) LPS blasts (2.5 x 106) produced from syngeneic unimmunized mice. Ag-pulsed LPS blast cells consisted of splenocytes that have been cultured in RPMI 1640, in the presence of 25 µg/ml LPS (Sigma-Aldrich), supplemented with 10% FCS, 5 x 10–5 M 2-ME, 1 mM glutamine, 1 mM sodium pyruvate, and 7 µg/ml dextran sulfate (Sigma-Aldrich) for 3 days and pulsed for 3 h with 50 µg/ml of the same bacteriophage preparations used for the immunization procedure. After 5–6 days of coculture, effector cells were harvested and assayed for cytotoxic activity and pentamer staining.

Proliferation assay

T cell lines (2 x 104 cells/well) were incubated with p23-pulsed irradiated autologous APCs (5 x 104 cells/well) for 36 h at 37°C, pulsed with [3H]thymidine (0.5 µCi/well, specific activity 5 Ci/mmol; Amersham, GE Healthcare) for 12 h at 37°C, and harvested with a microplate cell harvester and counted in a TopCount β-counter (Packard). All experimental points were performed in triplicate and results are expressed as cpm ± SD.

Cytotoxic assay

Effector cell-mediated cytotoxic activity was evaluated by the standard 51Cr-release cytotoxicity assay. Briefly, target cell lines were labeled with 51Cr (Amersham, GE Healthcare) for 90 min at 37°C. Peptide-loaded target cells were obtained by pulsing 1 x 106 of T2 or RMA-S-HHD cells with 20 µg/ml MAGE-10254–262 or MAGE-3271–279 synthetic peptides for 2 h at 37°C. Effector cells were incubated with 5 x 103 target cells, at the indicated E:T ratios, for 5 h at 37°C. For Ab-blocking experiments, tumor target cells were treated with saturating concentrations of anti-HLA-A2.1 BB7.2 or irrelevant anti-HLA-DR L243 mAbs for 30 min at 37°C before incubation with effector cells. The percentage of specific cytotoxicity was calculated as 100 x (cpm experimental release – cpm spontaneous release)/(cpm maximum release – cpm spontaneous release). Spontaneous release was always ≤20%. All experimental points were performed in triplicate.

Pentamer staining and flow cytometric analysis

Cells were stained with PE-labeled HLA-A2/MAGE-A3271–279 (FLWGPRALV) pentamers (Proimmune) for 45 min at 4°C before addition of FITC-conjugated anti-human or anti-mouse CD8 mAbs (Proimmune). For nonspecific staining HLA-A2/RT2 (ILKEPVHGV) pentamers were used. Stained cells were analyzed by flow cytometry using a FACSCanto (BD Biosciences).

In vivo tumor protection assay

Eight- to 10-wk-old HHD mice were injected two times s.c. at the base of the tail at 3-wk intervals with 140 µg of double-display fd23/Mg3 virions or, as controls, with 140 µg of fd23/GAG virions or PBS. One week after the second injection, mice were challenged s.c. in the right flank with 5 x 104 EL-4-HHD/MAGE-A3 tumor cells (36). Tumor size was measured twice a week using calipers in the two perpendicular diameters. Tumor volume (mm3) was calculated using the equation (D x d2)/2, where d is the smaller of the two diameters. Animal survival was recorded until 80 days and, at the end of this period, tumor-free mice were classed as survivors.

Statistical analyses

All statistical analyses were performed with GraphPad Prism 4 software. Differences in tumor incidence and survival were evaluated by the Mantel-Haenszel log-rank test. Differences in tumor sizes were evaluated by the one-way ANOVA test, using the nonparametric Kruskal-Wallis and the Dunn’s multiple comparison tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Construction of double-hybrid bacteriophage virions

We constructed and purified two double-display fd23/Mg10 and fd23/Mg3 bacteriophage virions, coexpressing the promiscuous HLA-DR-restricted Th cell peptide p23, as well as the HLA-A2-restricted CTL peptide MAGE-A10254–262 or MAGE-A3271–279, respectively, on the same capsid. Based on the relative yields of various N-terminal sequences obtained by sequence analysis of the purified virions, we calculated that the number of copies of the major coat protein pVIII displaying p23 peptide was {cong}8% of the 2700 pVIII proteins per virion, while the number of copies of pVIII displaying MAGE-A10254–262 or MAGE-A3271–279 peptides was {cong}4% (for each peptide) of the 2700 pVIII proteins per virion.

Generation of MAGE-A10254–262 peptide-specific CTLs in vitro

To investigate the ability of double-display bacteriophage virions to induce peptide-specific CTL responses in vitro, PBMCs from three different HLA-A2+ healthy donors were stimulated with fd23/Mg10 virions, and effector cell-mediated cytotoxic activity toward HLA-A2+/MAGE-A10254–262 peptide-pulsed target cells was examined. Autologous adherent PBMCs, used as APCs, were pulsed with 50 µg/ml fd23/Mg10 bacteriophage preparations or, as a control, with 50 µg/ml wild-type bacteriophage fd in the presence of 1.2 µg/ml p23 and 0.35 µg/ml MAGE-A10254–262 synthetic peptides. The concentrations of both synthetic peptides corresponded to those of the two peptides expressed on fd23/Mg10 virion capsids. The generation of MAGE-A10254–262 peptide-specific CTLs was assessed in a 51Cr-release assay using relevant or irrelevant peptide-loaded T2 target cell lines. As shown in Fig. 1A, comparable levels of specific cytotoxic activity were exerted by all different T cell lines generated by stimulation with fd23/Mg10-pulsed APCs, whereas no cytotoxicity was observed when T cell lines were raised with APCs pulsed with wild-type bacteriophage fd in the presence of p23 and MAGE-A10254–262 synthetic peptides. Cytotoxic activity toward peptide-unloaded T2 cells was always ≤10% (data not shown). Moreover, according to previous results obtained using fd virions engineered to display HIV-1-RT peptides (29), no cytotoxic activity was observed when PBMCs were stimulated with single-display fdMg10 virions (data not shown), indicating that Th cells were necessary for the induction of CTL responses. In this regard, we observed that the three independent healthy donors displayed comparable proliferative responses to the p23 synthetic helper peptide (Fig. 1B). Taken together, these results showed that, in contrast to stimulation with low doses of synthetic peptides, stimulation with double-display bacteriophage virions, displaying the same amount of peptides, elicited TAA peptide-specific CTL responses in vitro. These data indicated that engineered fd virions were able to enhance the immunogenicity of delivered peptides.


Figure 1
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  		FIGURE 1. Generation of human peptide-specific CTLs by double-hybrid fd23/Mg10 bacteriophages in vitro. T cell lines were derived from three different HLA-A2+ healthy donors (LDA, ACA, and ACN) by coculturing nonadherent PBMCs with autologous adherent PBMCs (used as APCs) pulsed with 50 µg/ml fd23/Mg10 (filled symbols) or, as a control, with 50 µg/ml wild-type bacteriophage fd in the presence of 1.2 µg/ml p23 and 0.35 µg/ml MAGE-A10254–262 synthetic peptide (open symbols). Growing T cell lines were boosted using the conditions mentioned above. Nine days after the second stimulation (A), effector cell-mediated cytotoxicity was assayed toward relevant (loaded with MAGE-A10254–262 peptide: {blacksquare}, {square}) or irrelevant (loaded with MAGE-A3271–279 peptide: {diamondsuit}, {diamond}) T2 (Tap–/–, HLA-A2.1+) target cells. B, Proliferative responses of T cell lines from donors LDA (black bars), ACA (gray bars), and ACN (white bars) were assayed toward autologous irradiated APCs pulsed with different concentrations of p23 synthetic peptide. Cells were pulsed with [3H]thymidine, all experimental points were performed in triplicate, and results are expressed as cpm ± SD. C, Effector cell-mediated cytotoxity was assayed toward a panel of tumor target cell lines (described in Table I). Data are representative of at least two different experiments performed with T cell lines from each donor.

 
Recognition of naturally processed MAGE-A10 Ag on tumor cells by MAGE-A10254–262 peptide-specific CTLs

To investigate whether double-display fd23/Mg10 virions were able to induce the generation of MAGE-A10254–262 peptide-specific CTLs capable of recognizing the endogenously processed peptide on the surface of HLA-A2+/naturally expressing MAGE-A10 tumor cells, CTL-mediated cytotoxic activity toward a panel of tumor target cell lines (Table I) was examined. As shown in Fig. 1C, significant cytotoxic activity was induced against HLA-A2+/MAGE-A10+ J82 and SD48 bladder TCC as well as 10538 melanoma target cells. In contrast, no lysis of HLA-A2+/MAGE-A10– 1512 TCC cells and HLA-A2–/MAGE-A10+ T24 and M14 tumor cell lines was observed (Fig. 1C). Moreover, according to the data obtained using peptide-loaded target cells, no cytotoxic activity was generated when effector cells were raised with APCs pulsed with 50 µg/ml wild-type bacteriophage fd in the presence of 1.2 µg/ml p23 and 0.35 µg/ml MAGE-A10254–262 synthetic peptides or pulsed with single-display fdMg10 virions (data not shown). These results showed that stimulation with double-display bacteriophage virions was very effective in inducing the generation of peptide-specific CTLs capable of recognizing naturally expressing TAAs on tumor cells.

Generation of MAGE-A3271–279 peptide-specific CTLs capable of recognizing naturally processed MAGE-A3 Ag on tumor cells

To examine the strength of the antitumor CTL response elicited by double-display bacteriophages, we examined the ability of double-hybrid fd23/Mg3 virions to induce the generation of CTLs specifically recognizing the MAGE-A3271–279-peptide, whose natural expression level on the neoplastic cell surface has been reported to be very low (13, 22, 23). To this purpose, PBMCs from the three different HLA-A2+ healthy donors were stimulated with APCs pulsed with 50 µg/ml fd23/Mg3 virions or, as a control, with APCs pulsed with 50 µg/ml wild-type bacteriophage fd in the presence of 1.2 µg/ml p23 and 0.35 µg/ml MAGE-A3271–279 synthetic peptides. Effector cell-mediated cytotoxic activity toward HLA-A2+/MAGE-A3271–279 peptide-pulsed and HLA-A2+/MAGE-A3+ tumor target cell lines (Table I) was examined.

We observed that effector cells derived from different PBMCs stimulated with fd23/Mg3 exerted comparable levels of cytotoxic activity not only toward HLA-A2+/peptide-pulsed T2 cells (Fig. 2A), but also, most remarkably, toward HLA-A2+/MAGE-A3+ 1512 and SD48 bladder TCC cell lines (Fig. 2B). In contrast, T cell lines induced by stimulation with APCs pulsed with wild-type bacteriophage fd in the presence of p23 and MAGE-A3271–279 synthetic peptides were not able to kill MAGE-A3271–279 peptide-pulsed (Fig. 2A) or MAGE-A3+ tumor target cells (data not shown). We thus investigated the levels of HLA-A2 protein expression using the anti-HLA-A2.1 mAb and the levels of MAGE-A3 transcript expression on relevant tumor target cells. As illustrated in Fig. 3A, different expression levels of HLA-A2 surface protein as well as different amounts of MAGE transcripts (Fig. 3B) were observed among the different tumor cell lines. The data illustrated in Fig. 3 indicated that an inverse correlation existed between HLA-A2 and MAGE-A3 expression levels in the different tumor target cells. This finding, together with the fact that similar levels of Th responses were displayed by the three different donors (Fig. 1B), might explain why the different effector T cells, obtained by stimulation with fd23/Mg3 virions, exerted comparable levels of lytic activity toward the different tumor target cells. To further verify the specificity of Ag recognition by CTLs, cold target-cell inhibition assays were performed by adding unlabeled MAGE-A3271–279 peptide-pulsed T2 cells to labeled 1512 and SD48 target cell lines. As shown in Fig. 2C, peptide-loaded T2 cells inhibited effector cell-mediated cytotoxicity toward 1512 and SD48 target cells, whereas no inhibition was observed when cold peptide-unloaded T2 cells were added to the assay. We also ascertained that specific tumor cell recognition by CTLs was HLA-A2 restricted, because pretreatment of relevant target cells with the anti-HLA-A2.1 mAb inhibited lymphocyte-mediated target cell killing; in contrast, no inhibition was found when target cells were pretreated with the irrelevant anti-HLA-DR mAb (Fig. 2C).


Figure 2
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  		FIGURE 2. Generation of human MAGE-A3271–279 peptide-specific CTLs in vitro recognizing HLA-A2+/MAGE-A3+ tumor cells. T cell lines were derived from three different HLA-A2+ healthy donors (LDA, ACA, ACN) by coculturing nonadherent PBMCs with autologous APCs pulsed with 50 µg/ml fd23/Mg3 (filled symbols) or, as a control, with 50 µg/ml wild-type bacteriophage fd in the presence of 1.2 µg/ml p23 and 0.35 µg/ml MAGE-A3271–279 synthetic peptides (open symbols). Growing T cell lines were boosted using the conditions mentioned above. Nine days after the second stimulation, effector cell-mediated cytotoxicity was assayed toward: (A) relevant (loaded with MAGE-A3271–279 peptide: {diamondsuit}, {diamond}) or irrelevant (loaded with MAGE-A10254–262 peptide: {blacksquare}, {square}) T2 (Tap–/–, HLA-A2.1+) target cells; (B) a panel of tumor target cell lines (Table I). C, Relevant tumor target cell lines are indicated in the abscissa (black bars); shown also are target cells pretreated with the anti-HLA-A2.1 BB7.2 mAb (white bars) or the irrelevant anti-HLA-DR mAb (dark gray bars), and target cells in the presence of unlabeled MAGE-A3271–279 peptide-loaded (hatched bars) or unloaded (light gray bars) T2 cells (1:90 hot/cold target cell ratio). E:T ratios (30:1) are shown and the means ± SD of triplicates are indicated. Cytotoxic activities were evaluated in a 5-h 51Cr-release assay. Data are representative of at least two different experiments.

 

Figure 3
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  		FIGURE 3. HLA-A2 protein and MAGE-A3 transcript expression levels in HLA-A2+/MAGE-A3+ tumor cell lines. A, HLA-A2 membrane expression was assessed by cytofluorimetry using anti-HLA-A2 BB7.2 (right panel) or irrelevant isotype control (left panel) mAbs in the indicated tumor cell lines. The percentages of positive cells are shown in the right upper quadrants. B, MAGE-A3 mRNA was analyzed by real-time PCR. Relative MAGE-A3 mRNA levels were calculated by the comparative cycle threshold (CT) method and normalized by GAPDH expression. Results are the means ± SD of three independent experiments normalized to SK23-MEL expression.

 
To directly visualize Ag-specific CD8+ T cells, effector T cell populations derived from the three donors were stained with anti-CD8 mAb and HLA-A2/MAGE-A3271–279 pentamers. As shown in Fig. 4, the expansion of a MAGE-A3271–279-specific CD8+ T cell population was detected in all different T cell lines induced by fd23/Mg3-pulsed APCs. In contrast, no expansion of specific CD8+ T cells was observed when T cell lines were induced by APCs pulsed with wild-type bacteriophage fd in the presence of p23 and MAGE-A3271–279 synthetic peptides (Fig. 4).


Figure 4
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  		FIGURE 4. Pentamer analysis in human MAGE-A3271–279 peptide-specific CTLs. Effector T cell populations (from donors LDA, ACA, and ACN) raised with fd23/Mg3-pulsed APCs were stained with FITC-conjugated anti-CD8 mAb and irrelevant PE-conjugated HLA-A2/RT2 pentamers (left panel) or HLA-A2/MAGE-A3271–279 pentamers (right panel). Flow cytometric analysis was performed using FACSCanto. Pentamer staining is also reported of a representative effector T cell line population raised with APCs pulsed with wild-type bacteriophage fd in the presence of p23 and MAGE-A3271–279 synthetic peptides. The numbers represent the percentage of pentamer-positive cells after gating on CD8+ cells. Data are representative of at least two different experiments.

 
To assess whether our results could be related to the histological origin of the tumor cell lines used as target cells, we examined whether CTLs were also capable of killing the HLA-A2+/MAGE-A3+ SK23-MEL melanoma cell line, where an impaired MAGE-A3271–279 peptide processing has been previously described (13, 22, 23). As shown in Fig. 5, effector cells were capable of exerting cytotoxic activity against SK23-MEL tumor cells. To verify the specificity of peptide recognition by CTLs, cold target-cell inhibition assays were performed by adding excess concentrations of unlabeled MAGE-A3271–279 peptide-pulsed T2 cells to labeled SK23-MEL target cells. As shown in Fig. 5, a 90-fold higher concentration of peptide-loaded T2 cells significantly inhibited effector cell-mediated cytotoxicity toward SK23-MEL target cell lines. In contrast, a 90-fold higher concentration of peptide-unloaded T2 cells did not modify the cytotoxic activity (Fig. 5). We then examined whether treatment of SK23-MEL cells with lactacystin, a specific proteasome inhibitor that has been reported to restore MAGE-A3271–279 peptide presentation on SK23-MEL tumor cells (22), could modify CTL-mediated cytotoxic activity. In contrast to results obtained with lactacystin treated HLA-A2+/MAGE-A3– J82 target cells, a significant enhancement of effector cell-mediated cytotoxicity was observed when SK23-MEL cells were treated with lactacystin (Fig. 5). We also established that the specific tumor cell recognition was HLA-A2 restricted; indeed, pretreatment of SK23-MEL target cells with anti-HLA-A2.1 mAbs inhibited lymphocyte-mediated killing, whereas no inhibition was observed when target cells were pretreated with the irrelevant anti-HLA-DR mAb (Fig. 5). Taken together, these results showed that specific CTLs generated by engineered virions were able to recognize tumor cells expressing low amounts of surface peptide-HLA epitope complexes, indicating that this peptide delivery system could elicit potent antitumor CTL responses.


Figure 5
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  		FIGURE 5. Recognition of naturally processed MAGE-A3271–279 peptide on human SK23-MEL melanoma cells. The cytotoxicity of MAGE-A3271–279 peptide-specific CTLs was evaluated toward HLA-A2+/MAGE-A3+ SK23-MEL or, as a control, J82 HLA-A2+/MAGE-A3– tumor target cells in a 5-h 51Cr-release assay at the indicated E:T ratios. For assessment of peptide-specific recognition by CTLs, increased excess concentrations of unlabeled MAGE-A3271–279 peptide-loaded T2 cells were added to labeled SK23-MEL target cells. Unlabeled peptide-unloaded T2 cells were used as a control. For proteasome inhibition experiments, SK23-MEL or J82 target cells were treated with 10 µM of lactacystin for 17 h at 37°C before the cytotoxicity assay. For assessment of HLA-A2-restricted recognition by CTLs, SK23-MEL target cells were pretreated with the anti-HLA-A2.1 BB7.2 mAb or the irrelevant anti-HLA-DR mAb. Data are representative of three different experiments. The means ± SD of triplicates are indicated.

 
Isolation of high-avidity MAGE-A3271–279 peptide-specific CD8+ CTL clones in vitro

To further characterize the antitumor CTL response elicited by double-display bacteriophages, we isolated CD8+ T lymphocytes from T cell lines (derived from LDA donor) that had been stimulated with double-hybrid fd23/Mg3 virions, which we cloned by limiting dilution, and we analyzed their specificity and their functional avidity. Approximately 200 CD8+ T cell clones were obtained and screened for their cytotoxic activity toward the HLA-A2+/MAGE-A3+ 1512 target cell line in a 51Cr-release assay. Specific cytotoxic activity against the 1512 target cell line was exerted by two clones, named 8 and Y (Fig. 6, inset). Moreover, tumor cell killing was specific and HLA-A2 restricted, because the cytotoxic activities exerted by the two clones toward 1512 target cells were inhibited by the unlabeled peptide-pulsed T2 cell line as well as by the anti-HLA-A2 mAb (Fig. 6, inset). Note that all the other clones did not exert any lytic activity toward peptide-pulsed T2 target cells. Additionally, clones 8 and Y were positive for HLA-A2/MAGE-A3271–279 pentamer staining (data not shown). To characterize the avidity of the specific CD8+ CTL clones, we tested their ability to kill T2 target cells loaded with decreasing concentrations of MAGE-A3271–279 peptide, from 107 to 10 pg/ml. Both CD8+ T cell clones were able to kill T2 target cells loaded with as little as 103 pg/ml of specific peptide (Fig. 6), showing their high avidity for Ag-specific recognition.


Figure 6
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  		FIGURE 6. Analysis of human MAGE-A3271–279-specific CD8+ CTL clones. The avidity of MAGE-A3271–279-specific CD8+ CTL clones was assessed analyzing the cytotoxic activity exerted by CTL clones, 8 ({triangleup}) and Y ({blacksquare}), toward T2 target cells loaded with decreasing concentrations (from 107 to 10 pg/ml) of MAGE-A3271–279 synthetic peptide. Inset, Cytotoxicity toward: the HLA-A2+/MAGE-A3+ 1512 tumor cell line (black bars); 1512 cells in the presence of unlabeled (1:90 hot/cold target ratio) MAGE-A3271–279 peptide-loaded T2 cells (white bars); and 1512 target cells pretreated with the anti-HLA-A2.1 BB7.2 mAb (gray bars). Cytotoxicity was evaluated in a 5-h 51Cr-release assay. E:T ratios (30:1) are shown and the means ± SD of triplicates are indicated.

 
Generation of MAGE-A10254–262 and MAGE-A3271–279 peptide-specific CTLs in vivo

To investigate the ability of double-display bacteriophage virions to elicit peptide-specific CTL responses in vivo, HHD (HLA-A2.1+/H2-Db+) transgenic mice were immunized with double-display fd23/Mg10 or fd23/Mg3 virions, and the generation of specific CTL activities toward peptide-pulsed target cell lines was examined. Mice were injected s.c. with 140 µg of fd23/Mg10 or fd23/Mg3 bacteriophage preparations or, as a control, with 140 µg/ml wild-type bacteriophage fd in the presence of 3.4 µg of p23 and 0.98 µg of MAGE-A10254–262 or MAGE-A3271–279 synthetic peptides. As for the in vitro studies, all synthetic peptides were used at the same concentrations as those of peptides expressed on double-display fd23/Mg10 and fd23/Mg3 virion capsids. Splenocytes were isolated and restimulated in vitro with syngeneic LPS-induced blast cells pulsed with the same bacteriophage virions used for the immunization. Effector cell-mediated cytotoxic activities were tested in 51Cr-release assays toward RMA-S-HHD (Tap–, HLA-A2.1+) target cell lines loaded with MAGE-A10254–262 or MAGE-A3271–279 synthetic peptides. As shown in Fig. 7, A and B, specific cytotoxic activities were generated in splenocytes isolated from mice immunized with fd23/Mg10 or fd23/Mg3 virions. In contrast, no cytotoxic activity was found in splenocytes isolated from mice immunized with wild-type bacteriophage fd in the presence of p23 and MAGE-A10254–262 or MAGE-3271–279 synthetic peptides (Fig. 7, A and B). Splenocytes isolated from unimmunized mice and restimulated in vitro with fd23/Mg10- or fd23/Mg3-pulsed LPS blasts did not exert cytotoxic activities (data not shown). We also observed the ability of splenocytes isolated from mice immunized with fd23/Mg3 virions to kill the murine EL-4-HHD/MAGE-A3 tumor cell line (Fig. 7C), whereas they did not kill human HLA-2+/MAGE-A3+ 1512 tumor cells (data not shown), indicating that recognition by CTLs was CD8 coreceptor dependent. The expansion of MAGE-A3271–279 Ag-specific CD8+ T cells was also demonstrated by positive staining of splenocytes with HLA-A2/MAGE-A3271–279 pentamers (Fig. 7D). These results showed that the double-display bacteriophage delivery system was very effective in eliciting peptide-specific CTL responses in vivo.


Figure 7
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  		FIGURE 7. Generation of peptide-specific CTLs by double-hybrid bacteriophage immunizations in HHD mice. HHD (HLA-A2.1+/H-2Db+) transgenic mice were immunized twice with 140 µg of fd23/Mg10 or of fd23/Mg3 virions or, as a control, with 140 µg/ml wild-type bacteriophage fd in the presence of 3.4 µg/ml p23 and 0.98 µg/ml MAGE-A10254–262 or MAGE-A3271–279 synthetic peptides (as indicated in abscissae). Splenocytes were restimulated in vitro with syngeneic LPS blasts pulsed with double-hybrid bacteriophages. Effector cell-mediated cytotoxic activities were evaluated toward (A) Mg10 and (B) Mg3 peptide-loaded RMA-S-HHD target cells. Cytotoxic acitivities were evaluated in a 5-h 51Cr-release assay. The means ± SD of 12 mice/experimental group are indicated. C, Cytotoxicity exerted by effector cells obtained from mice immunized with fd23/Mg3 ({blacksquare}), fd23/Mg10 (x), or wild-type bacteriophage fd in the presence of synthetic peptides ({triangleup}) was assayed toward EL-4-HHD/MAGE-A3 tumor target cells (a representative experiment is reported). D, Pentamer analysis of splenocytes obtained from mice immunized with PBS (left panel) or with fd23/Mg3 virions (right panel). Splenocytes were stained with FITC-conjugated anti-CD8 mAb and PE-conjugated HLA-A2/MAGE-A3271–279 pentamers. Flow cytometric analysis was performed using FACSCanto. The numbers in the upper right panel represent the percentage of pentamer-positive cells after gating on CD8+ cells. Data are representative of at least three different experiments.

 
Induction of tumor protection in vivo

To assess whether double-display fd23/Mg3 virions elicited an effective in vivo antitumor immune response, HHD mice were primed with fd23/Mg3 hybrid phages once and then boosted after 3 wk. HHD mice injected with the fd23/GAG irrelevant hybrid bacteriophages or PBS were used as controls. Seven days after the second immunization, mice were challenged with EL-4-HHD/MAGE-A3 tumor cells, and the incidence and growth of tumor, as well as animal survival, were monitored.

At day 17 after tumor challenge, a palpable tumor appeared in 67% of mice inoculated with PBS, and in 8 and 21% of mice immunized with fd23/GAG or fd23/Mg3 hybrid bacteriophage particles, respectively (Fig. 8A). At day 21, a tumor was present in 100% of mice injected with PBS, and in 52 and 36% of fd23/GAG- or fd23/Mg3-immunized mice, respectively (Fig. 8A). At day 28, a tumor was present in 100% of fd23/GAG-immunized mice, whereas 60% of mice vaccinated with the fd23/Mg3 virions carried a tumor; this observation remained unchanged until day 80 (Fig. 8A). Statistical analysis indicated that protection observed in mice vaccinated with fd23/Mg3 virions was significant (p = 0.0003, by the Mantel-Haenszel log-rank test). Therefore, a significant lower tumor incidence, with up to 40% tumor-free animals at the end of the experiment, was evident in the fd23/Mg3-vaccinated mice as compared with the controls. Note that the lack of protection in 60% of mice vaccinated with fd23/Mg virions was not dependent on the loss of one or both genes in the progressive lesions. Indeed, the expressions of both gene products, the HLA-A2 protein and the MAGE-A3 transcript, were still observed in EL-4-HHD/MAGE-A3-derived tumors isolated from mice vaccinated with fd23/Mg3 hybrid phages (Fig. 8, C and D).


Figure 8
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  		FIGURE 8. Tumor growth prevention by vaccination with fd23/Mg3 virions in HHD mice. HHD mice were immunized twice with fd23/Mg3 virions, fd23/GAG irrelevant virions, or PBS at days 21 and 7 before tumor challenge (day 0) with EL-4-HHD/MAGE-A3 tumor cell lines. A, Percentages of tumor-free mice. Tumor protection in mice vaccinated with fd23/Mg3 virions was significant (p = 0.0003, by the Mantel-Haenszel log-rank test). B, Tumor volumes recorded 30 days after tumor challenge; the lines represent the median values. Prevention of tumor growth in mice vaccinated with fd23/Mg3 virions was significant (p < 0.0001, by the one-way ANOVA Kruskal-Wallis test). Each experimental group contained 5–10 mice. Cumulative results of at least three independent experiments are shown. EL4-HHD/MAGE-A3 cell line-derived tumors were isolated from HHD mice immunized with fd23/Mg3 hybrid phages, fd23/GAG virions, or PBS, and (C) Western blot analysis of HLA-A2 expression, using the anti-HLA-A2 HCA2 mAb, and (D) RT-PCR analyses of MAGE-A3 expression were performed. The EL-4-HHD/MAGE-A3 and the EL-4 tumor cell lines were used as positive and negative controls, respectively. A representative experiment is reported. Actin is used as expression control.

 
The analysis of tumor growth was also performed and, as shown in Fig. 8B, a significant prevention of tumor growth was observed in mice vaccinated with fd23/Mg3 virions (p < 0.0001, by the one-way ANOVA Kruskal-Wallis test). It is noteworthy that immunization with fd23/GAG phages did exhibit modest antitumor effects, although they were not significant (p > 0.05, by the one-way ANOVA using the Dunn’s multiple comparison test as a posttest) (Fig. 8, A and B). Moreover, one stable disease and two complete tumor regressions were observed in fd23/Mg3-immunized mice. Therefore, at the end of the experiment (day 80 after tumor challenge), a 60% survival rate was evaluated for fd23/Mg3-immunized mice, whereas all fd23/GAG- or PBS-injected mice died at days 60 and 34, respectively (data not shown). Overall, these results showed that fd23/Mg3 hybrid vaccination was efficient at hampering tumor growth.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we describe a novel delivery system for TAA-derived peptides based on nonpathogenic, engineered filamentous bacteriophage fd, and we document its ability to induce potent antitumor CTL responses in a human cell system in vitro and in a humanized murine model in vivo. The fact that the stimulation with wild-type bacteriophage fd in the presence of the Th and CTL synthetic peptides, used at the same concentrations as those of peptides expressed on the virion capsides, was incapable of generating cytotoxic activities both in vitro and in vivo indicated that engineered bacteriophages were capable of increasing substantially the immunogenicity of the delivered peptides. The strength of this peptide delivery system was further supported by the unexpected ability of MAGE-A3271–279 peptide-specific CTLs to kill tumor cells that have been described to express low amounts of MAGE-A3271–279 peptide-HLA epitope complexes on the their cell surface (22, 23), and that are not usually recognized by peptide-specific CTLs generated by conventional repeated stimulation procedures (13). Therefore, our results indicate that the fd virion peptide delivery system may overcome the limitation reported in conventional peptide vaccinations for the generation of specific CTLs directed toward tumor cells expressing scarce surface peptide-HLA epitope complexes. The mechanisms underlying the efficacy of engineered virions to generate specific and potent antitumor CTLs are currently unknown and are under investigation in our laboratory. It has been previously described by others and by us that filamentous bacteriophage fd are taken up and processed by MHC class I and class II pathways (38, 39). This might explain, at least in part, the ability of virions displaying foreign T cell epitopes to elicit potent Th cell-dependent CTL responses. Additionally, we herein described that fd23/Mg3 virions were capable of generating high-avidity CTL clones in vitro. Moreover, the induction of CD8+ effector cells recognized by HLA-A2/MAGE-A3271–279 pentamer in mice immunized with fd23/Mg3 phage particles was also shown. It is noteworthy that tetramer analysis on effector cells derived from HHD mice performed using HLA-A2 instead of HHD peptide tetramers has been previously described to allow the exclusive recognition of Ag-specific high-avidity CD8+ T cells (40, 41). Therefore, the detection in our system of CD8+ CTLs capable of binding HLA-A2/MAGE-A3271–279 pentamers indicated the generation of Ag-specific high-avidity CTLs in vivo. Thus, from these results, we can speculate that an efficient processing of engineered bacteriophages by APCs enhances substantially the immunogenicity of peptides, which results in the generation of high-avidity CTLs capable of recognizing tumor cells expressing low amounts of surface peptide-HLA epitope complexes.

The in vivo antitumor efficacy of the phage delivery system was indicated by the induction of tumor protection in HHD mice vaccinated with fd23/Mg3 phage particles. Note that bacteriophages have been safely administered in humans and their efficacy has been shown, even if their administration induced the production of anti-bacteriophage Abs by the host (42, 43, 44).

We ruled out that the generation of potent antitumor CTLs by engineered bacteriophage virions was due to the presence of contaminants within the bacteriophage preparations, because wild-type and double-display virions were obtained using the same experimental procedures, and the same results were found in experiments performed using different preparations of both wild-type and double-display bacteriophages.

Overall, our results indicate that engineered bacteriophages represent a very effective TAA-derived peptide delivery system, capable of inducing potent CTL responses. Indeed, it is known that the generation of specific CTLs induced by antigenic peptide delivered by strongly immunogenic carriers, such as plasmidic DNA, replication-defective adenovirus, or dendritic cells, usually requires repeated stimulations in vitro as well as in vivo (14, 17, 18, 19, 45). However, future work will be performed to compare the strength of the fd virion delivery system with other systems, such as those mentioned above.

In conclusion, we herein describe that the delivery of TAA-derived peptides via nonpathogenic filamentous bacteriophage fd virions induces potent specific CTLs, thus representing a novel powerful system for the development of more effective peptide-based cancer vaccines.


   Acknowledgments
 
We thank Prof. M. C. Mazzilli, Dr. B. Mora, and Dr. D. D’Eliseo for DNA and protein HLA typing, and G. Bertini and P. Piccoli for husbandry of mice and technical assistance. We also thank P. Barba for helpful discussions and technical assistance. We thank Prof. G. Forni for helpful advice on mice experiments and data analysis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
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.

1 This work was supported by European Community (QLK3-CT-1999-00064), Italian Ministry of University and Research and Tuscia University, and "Fondo per gli Investimenti della Ricerca di Base" (RBLA033WJX). Back

2 F.V. and P.D.B. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Piergiuseppe De Berardinis, Institute of Protein Biochemistry, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, Naples 80131, Italy. E-mail address: p.deberardinis{at}ibp.cnr.it Back

4 Abbreviations used in this paper: TAA, tumor-associated Ag; CT, comparative cycle threshold; TCC, transitional-cell carcinoma. Back

Received for publication July 26, 2007. Accepted for publication January 9, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. van der Bruggen, P., C. Traversari, P. Chomez, C. Lurquin, E. De Plaen, B. van den Eynde, A. Knuth, T. Boon. 1991. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 254: 1643-1647. [Abstract/Free Full Text]
  2. Boon, T., P. van der Bruggen. 1996. Human tumor antigens recognized by T lymphocytes. J. Exp. Med. 183: 725-729. [Free Full Text]
  3. Wang, R. W., S. A. Rosenberg. 1999. Human tumor antigens for cancer vaccine development. Immunol. Rev. 170: 85-100. [Medline]
  4. Scanlan, M. J., A. O. Gure, A. A. Jungbluth, L. J. Old, Y. T. Chen. 2002. Cancer/testis antigens: an expanding family of targets for cancer immunotherapy. Immunol. Rev. 188: 22-32. [Medline]
  5. De Plaen, E., K. Arden, C. Traversari, J. J. Gaforio, J.-P. Szikora, C. De Smet, F. Brasseur, P. van der Bruggen, B. Lethé, C. Lurquin, et al 1994. Structure, chromosomal localization, and expression of 12 genes of the MAGE family. Immunogenetics 40: 360-369. [Medline]
  6. Chomez, P., M. De Backer, E. Bertrand, T. De Plaen, T. Boon, S. Lucas. 2001. An overview of the MAGE gene family with the identification of all human members of the family. Cancer Res. 61: 5544-5551. [Abstract/Free Full Text]
  7. Rimoldi, D., S. Salvi, D. Reed, P. Coulie, V. C. Jongeneel, E. De Plaen, F. Brasseur, A.-M. Rodriguez, T. Boon, J. C. Cerottini. 1999. cDNA and protein characterization of human MAGE-10. Int. J. Cancer 82: 901-907. [Medline]
  8. van den Eynde, B., and P. van der Bruggen. 2001. Peptide database of T-cell defined tumor antigens. Cancer Immunity http://www.cancerimmunity.org/peptidedatabase/Tcellepitopes.htm
  9. van der Bruggen, P., Y. Zhang, P. Chaux, V. Stroobant, C. Panichelli, E. S. Schultz, J. Chapirovan, B. J. van den Eynde, F. Brasseur, T. Boon. 2002. Tumor-specific shared antigenic peptides recognized by human T cells. Immunol. Rev. 188: 51-64. [Medline]
  10. Imanishi, T., T. Akaza, A. Kimura, K. Tokunaga, T. Gojobori. 1992. Allele and haplotype frequencies for HLA and complement loci in various ethnic groups. K. Tsuji, and M. Aizawa, and T. Sasazuki, eds. HLA 1991: Proceedings of the 11th Histocompatibility Workshop and Conference 1065-1067. Oxford University Press, Oxford.
  11. van der Bruggen, P., J. Bastin, T. Gajewski, P. G. Coulie, P. Boel, C. De Smet, C. Traversari, A. Townsend, T. Boon. 1994. A peptide encoded by human gene MAGE-3 and presented by HLA-A2 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE-3. Eur. J. Immunol. 24: 3038-3043. [Medline]
  12. Huang, L. Q., F. Brasseur, A. Serrano, E. De Plaen, P. van der Bruggen, T. Boon, A. Van Pel. 1999. Cytolytic T lymphocytes recognize an antigen encoded by MAGE-A10 on a human melanoma. J. Immunol. 162: 6849-6854. [Abstract/Free Full Text]
  13. Valmori, D., D. Liénard, G. Waanders, D. Rimoldi, J. C. Cerottini, P. Romero. 1997. Analysis of MAGE-3-specific cytolytic T lymphocytes in human leukocyte antigen-A2 melanoma patients. Cancer Res. 57: 735-741. [Abstract/Free Full Text]
  14. Tjandrawan, T., D. M. Martin, M. J. Maeuer, C. Castelli, M. J. Lotze, W. J. Storkus. 1998. Autologous human dendriphages pulsed with synthetic or natural tumor peptides elicit tumor-specific CTLs in vitro. J. Immunother. 21: 149-157. [Medline]
  15. Fujie, T., F. Tanaka, M. Mori, K. Takesako, K. Sugimachi, T. Akiyoshi. 1997. Induction of antitumor cytotoxic T lymphocytes from the peripheral blood mononuclear cells of cancer patients using HLA-A2-restricted MAGE-3 peptide in vitro. Clin. Cancer Res. 3: 2425-2430. [Abstract/Free Full Text]
  16. Valmori, D., V. Dutoit, V. Rubio-Godoy, C. Chambaz, D. Liénard, P. Guillame, P. Romero, J. C. Cerottini, D. Rimoldi. 2001. Frequent cytolytic T-cell responses to peptide MAGE-A10254-262 in melanoma. Cancer Res. 61: 509-512. [Abstract/Free Full Text]
  17. Brossart, P., S. Wirth, G. Stuhler, V. L. Reichardt, L. Kanz, W. Brugger. 2000. Induction of cytotoxic T-lymphocyte responses in vivo after vaccinations with peptide-pulsed dendritic cells. Blood 96: 3102-3108. [Abstract/Free Full Text]
  18. Gajewski, T. F., F. Fallarino, A. Ashikari, M. Sherman. 2001. Immunization of HLA-A+ melanoma patients with MAGE-3 or MelanA peptide-pulsed autologous peripheral blood mononuclear cells plus recombinant interleukin 12. Clin. Cancer Res. 7: 895-901.
  19. Sadanaga, N., H. Nagashima, K. Mashino, K. Tahara, H. Yamagushi, M. Ohta, T. Fujie, F. Tanaka, H. Inoue, K. Takesako, et al 2001. Dendritic cell vaccination with MAGE peptide is a novel therapeutic approach for gastrointestinal carcinomas. Clin. Cancer Res. 7: 2277-2284. [Abstract/Free Full Text]
  20. Parmiani, G., C. Castelli, P. Dalerba, R. Mortarini, L. Rivoltini, F. M. Marincola, A. Anichini. 2002. Cancer immunotherapy with peptide-based vaccines: what have we achieved? Where are we going?. J. Natl. Cancer Inst. 94: 805-818. [Abstract/Free Full Text]
  21. Coulie, P. G., P. van der Bruggen. 2003. T-cell responses of vaccinated cancer patients. Curr. Opin. Immunol. 15: 131-137. [Medline]
  22. Valmori, D., U. Gileadi, C. Servis, P. R. Dunbar, J.-C. Cerottini, P. Romero, V. Cerundolo, F. Lévy. 1999. Modulation of proteosomal activity required for the generation of a cytotoxic T lymphocyte-defined peptide derived from tumor antigen MAGE-3. J. Exp. Med. 15: 895-906.
  23. Miconnet, I., C. Servis, J. C. Cerottini, P. Romero, F. Lévy. 2000. Amino acid identity and/or position determines the proteosomal cleavage of the HLA-A*0201-restricted peptide tumor antigen MAGE-3271-279. J. Biol. Chem. 275: 26892-26897. [Abstract/Free Full Text]
  24. De Berardinis, P., L. D’Apice, A. Prisco, M. N. Ombra, P. Barba, G. Del Pozzo, S. Petukhov, P. Malik, R. N. Perham, J. Guardiola. 1999. Recognition of HIV-derived B and T cell epitopes displayed on filamentous phages. Vaccine 17: 1434-1441. [Medline]
  25. Greenwood, J., A. E. Willis, R. N. Perham. 1991. Multiple display of foreign peptides on a filamentous bacteriophage: peptides from Plasmodium falciparum circumsporozoite protein as antigens. J. Mol. Biol. 220: 821-827. [Medline]
  26. Willis, A. E., R. N. Perham, D. Wraith. 1993. Immunological properties of foreign peptides in multiple display on a filamentous bacteriophage. Gene 128: 79-83. [Medline]
  27. Minenkova, O. O., A. A. Ilyichev, G. P. Kishchenko, V. A. Petrenko. 1993. Design of specific immunogens using filamentous phage as the carrier. Gene 128: 85-88. [Medline]
  28. Di Marzo Veronese, F., A. E. Willis, C. Boyer-Thompson, E. Appella, R. N. Perham. 1994. Structural mimicry and enhanced immunogenicity of peptide epitopes displayed on filamentous bacteriophage: the V3 loop of HIV-1 gp120. J. Mol. Biol. 243: 167-172. [Medline]
  29. De Berardinis, P., R. Sartorius, C. Fanutti, R. N. Perham, G. Del Pozzo, J. Guardiola. 2000. Phage display of peptide epitopes from HIV-1 elicits strong cytolytic responses. Nat. Biotechnol. 18: 873-876. [Medline]
  30. Manca, F., P. De Berardinis, D. Fenoglio, M. N. Ombra, G. Li Pira, D. Saverino, M. Autiero, L. Lozzi, L. Bracci, J. Guardiola. 1996. Antigenicity of HIV-derived helper determinants in the context of carrier recombinant proteins: effect on T helper cell repertoire selection. Eur. J. Immunol. 26: 2461-2469. [Medline]
  31. Malik, P., R. N. Perham. 1997. Simultaneous display of different peptides on the surface of filamentous bacteriophage. Nucleic Acids Res. 25: 915-916. [Abstract/Free Full Text]
  32. Malik, P., R. N. Perham. 1996. New vectors for peptide display on the surface of filamentous bacteriophage. Gene 171: 49-51. [Medline]
  33. Chopin, D., R. Barei-Moniri, P. Maille, M. A. Le Frere-Belda, B. Muscatelli-Groux, N. Merendino, L. Lecerf, A. Stoppacciaro, F. Velotti. 2003. Human urinary bladder transitional cell carcinomas acquire the functional Fas ligand during tumor progression. Am. J. Pathol. 162: 1139-1149. [Abstract/Free Full Text]
  34. Mecklenburg, I., D. Weckermann, A. Zippelius, A. Schoberth, S. Petersen, N. Prang, G. Riethmuller, P. Kufer. 2007. A multimarker real-time RT-PCR for MAGE-A gene expression allows sensitive detection and quantification of the minimal systemic tumor load in patients with localized cancer. J. Immunol. Methods 323: 180-193. [Medline]
  35. Salter, R. D., D. N. Howell, P. Cresswell. 1985. Genes regulating HLA class I antigen expression in T-B lymphoblast hybrids. Immunogenetics 21: 235-246. [Medline]
  36. Pascolo, S., N. Bervas, J. M. Ure, A. G. Smith, F. A. Lemonnier, B. Perarnau. 1997. HLA-A2.1-restricted education and cytolytic activity of CD8+ T lymphocytes from β2 microglobulin (β2m) HLA-A2.1 monochain transgenic H-2Db β2m double knockout mice. J. Exp. Med. 185: 2043-2051. [Abstract/Free Full Text]
  37. Stam, N. J., T. M. Vroom, P. J. Peters, E. B. Pastoors, H. L. Ploegh. 1990. HLA-A- and HLA-B-specific monoclonal antibodies reactive with free heavy chains in Western blots, in formalin-fixed, paraffin-embedded tissue sections and in cryo-immuno-electron microscopy. Int. Immunol. 2: 113-125. [Abstract/Free Full Text]
  38. Gaubin, M., C. Fanutti, Z. Mishal, A. Durrbach, P. De Berardinis, R. Sartorius, G. Del Pozzo, J. Guardiola, R. N. Perham, D. Piatier-Tonneau. 2003. Processing of filamentous bacteriophage virions in antigen-presenting cells targets both class I and class II peptide loading compartments. DNA Cell Biol. 22: 11-18. [Medline]
  39. Wan, Y., Y. Wu, J. Zhou, L. Zou, Y. Liang, J. Zhao, Z. Jia, J. Engberg, J. Bian, W. Zhou. 2005. Cross-presentation of phage particle antigen in MHC class II and endoplasmic reticulum marker-positive compartments. Eur. J. Immunol. 35: 2041-2050. [Medline]
  40. Hernandez, J., P. P. Lee, M. M. Davis, L. A. Sherman. 2000. The use of HLA A2.1/p53 tetramers to visualize the impact of self tolerance on the TCR repertoire. J. Immunol. 164: 596-602. [Abstract/Free Full Text]
  41. Choi, E. M., J. L. Chen, L. Wooldridge, M. Salio, A. Lissina, N. Lissin, I. F. Hermans, J. D. Silk, F. Mirza, M. J. Palmowski, et al 2003. High avidity antigen-specific CTL identified by CD8-independent tetramer staining. J. Immunol. 171: 5116-5123. [Abstract/Free Full Text]
  42. Ochs, H. D., R. H. Buckley, R. H. Kobayashi, A. L. Kobayashi, R. U. Sorensen, S. D. Douglas, B. L. Hamilton, M. S. Hershfield. 1992. Antibody responses to bacteriophage phi X174 in patients with adenosine deaminase deficiency. Blood 80: 1163-1171. [Abstract/Free Full Text]
  43. Sulakvelidze, A., Z. Alavidze, J. G. Morris, Jr. 2001. Bacteriophage therapy. Antimicrob. Agents Chemother. 45: 649-659. [Free Full Text]
  44. Clark, J. R., J. B. March. 2006. Bacteriophages and biotechnology: vaccines, gene therapy, and antibacterials. Trends Biotechnol. 24: 212-218. [Medline]
  45. Bullock, T. N., D. W. Mullins, V. H. T. Engelhard. 2003. Antigen density presented by dendritic cells in vivo differentially affects the number and avidity of primary, memory, and recall CD8+ T cells. J. Immunol. 170: 1822-1829. [Abstract/Free Full Text]




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