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The Journal of Immunology, 2006, 176: 217-224.
Copyright © 2006 by The American Association of Immunologists

Induction of Antitumor Immunity by CTL Epitopes Genetically Linked to Membrane-Anchored {beta}2-Microglobulin1

Alon Margalit2,*,{dagger}, Helena M. Sheikhet2,{ddagger}, Yaron Carmi3,*, Dikla Berko4,*, Esther Tzehoval{ddagger}, Lea Eisenbach{ddagger} and Gideon Gross5,*,{dagger}

* Laboratory of Immunology, MIGAL-Galilee Technology Center, Kiryat Shmona, Israel; {dagger} Department of Biotechnology, Tel-Hai Academic College, Upper Galilee, Israel; and {ddagger} Department of Immunology, Weizmann Institute of Science, Rehovot, Israel


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Level and persistence of antigenic peptides presented by APCs on MHC class I (MHC-I) molecules influence the magnitude and quality of the ensuing CTL response. We recently demonstrated the unique immunological properties conferred on APCs by expressing {beta}2-microglobulin ({beta}2m) as an integral membrane protein. In this study, we explored membrane-anchored {beta}2m as a platform for cancer vaccines using as a model MO5, an OVA-expressing mouse B16 melanoma. We expressed in mouse RMA-S cells two H-2Kb binding peptides from MO5, OVA257–264, and TRP-2181–188, each genetically fused with the N terminus of membranal {beta}2m via a short linker. Specific Ab staining and T cell hybridoma activation confirmed that OVA257–264 was properly situated in the MHC-I binding groove. In vivo, transfectants expressing both peptides elicited stronger CTLs and conferred better protection against MO5 than peptide-saturated RMA-S cells. Cells expressing OVA257–264/{beta}2m were significantly superior to OVA257–264-charged cells in their ability to inhibit the growth of pre-established MO5 tumors. Our results highlight the immunotherapeutic potential of membranal {beta}2m as a universal scaffold for optimizing Ag presentation by MHC-I molecules.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Most tumor-associated Ags (TAAs)6 known today were identified by their ability to induce cellular responses, predominantly mediated by CTLs. By virtue of their clonotypic TCR, CTLs recognize short peptides encased in a designated pocket formed by MHC class I (MHC-I) molecules, which are expressed by most nucleated cells in the body, including tumor cells. These consist of a membrane-attached, polymorphic H ({alpha})-chain, which functions as the peptide receptor, and {beta}2-microglobulin ({beta}2m), a noncovalently associated monomorphic L chain. CTL priming occurs in secondary lymphoid organs and requires presentation of antigenic peptides by MHC-I on professional APCs, primarily dendritic cells (DCs). The magnitude of CTL priming largely depends on the avidity of interactions between the TCR and the specific ligand presented by the DC. It is generally assumed that the priming step requires a higher density of MHC-I-peptide complexes on the APC than the activation of a fully differentiated, armed effector CTL by its target cell. In addition to Ag presentation, DCs provide necessary costimulatory signals and are capable of CTL induction via cross-presentation. Autologous DCs are, therefore, widely investigated as potential cancer vaccines (1).

APCs genetically modified to express preselected TAAs combine immunostimulatory activity with elevated and prolonged peptide presentation, and their efficacy in cancer immunotherapy is under intensive investigation (see Ref.2 for review). Gene delivery into APCs can be achieved by various experimental procedures. These include the use of an array of viral vectors, both ex vivo or in vivo; direct gene delivery, which is attained by several methods for DNA immunization (3, 4, 5, 6, 7); and the introduction of TAA-encoding mRNA ex vivo via relatively simple transfection procedures (8).

Following successful gene delivery into APCs, generation of CTL ligands from the gene product depends on the multistep MHC-I processing and presentation pathway within the cell (reviewed in Refs.9 and 10). A recent analysis described the overall efficacy of this pathway in quantitative molecular terms (11), concluding that an average of 2000 protein precursors were required to yield a single MHC-I complex at the cell surface. The density of such complexes, in turn, determines the degree of T cell responsiveness (12, 13, 14, 15, 16, 17). Increasing complex-precursor ratio is thus a major challenge in the design of genetic vaccines aiming at achieving optimal presentation for CTL induction. Targeting the nominal antigenic peptide directly to the endoplasmic reticulum (ER) circumvents proteasomal degradation, avoids rapid peptide loss due to excess proteolytic activity within the cytosol, and bypasses transportation to the ER by TAP (18, 19). Most attempts in this direction take advantage of the conventional leader peptide-mediated transportation of nascent polypeptides into the ER. The prevalent strategy makes use of minigenes, which tether the antigenic peptide directly to the C terminus of a leader peptide. Removal of the latter at the inner ER membrane liberates the final peptide, which can then diffuse freely within the ER lumen and compete with peptides from the native pool on binding to peptide-receptive MHC-I H-chain-{beta}2m heterodimers (20).

The prospects for binding are improved by genetically fusing such minigene products with the N terminus of an MHC-I component via a short linker. This has been accomplished by using the MHC-I H chain (21, 22), {beta}2m (23, 24), and {beta}2m-H-chain single chains, creating single-chain trimers (25, 26). Indeed, when these proteins were expressed on the surface of transfected cells, they were capable of conferring a tumor protection effect in syngeneic mice in vivo (21).

In a recent report (27), we described the creation of double-chimeric {beta}2m (dc{beta}2m)-based polypeptides. CTL epitopes, in the form of H-2Kk-restricted octapeptides, were linked to the N terminus of {beta}2m, whereas the C terminus of {beta}2m was bridged to the plasma membrane and supplemented with the transmembrane and cytoplasmic domains of the CD3 {zeta}-chain as a functional membranal anchor. When these constructs were expressed in transfected mouse T cells, ~20% of their surface H-2Kk molecules were occupied with the linked peptide. To study the net effect of membranal attachment of {beta}2m on the resulting MHC-I heterodimers, we explored a peptideless derivative of membranal {beta}2m in stably transfected RMA-S cells (28). Transfectants bound exogenous peptide considerably faster, and they did so at peptide concentrations 104- to 106-fold lower than parental cells, as detected by complex-specific Abs and by T cell activation. Membrane attachment of {beta}2m, therefore, appears to confer marked stabilization on resulting MHC-I molecules, which acquire a highly receptive state in RMA-S cells.

In this study, we evaluate membrane-anchored {beta}2m as a potentially universal backbone for maximizing MHC-I presentation of covalently linked peptides, using a mouse melanoma model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Vectors and expression plasmids

Chimeric {beta}2m genes were cloned into the mammalian expression vectors pBJ1-Neo (29) or pCI-Neo (Promega). An XbaI/BamHI stretch coding for mouse {beta}2m (m{beta}2m) leader peptide, the H-2Kb-binding antigenic peptide, and the N-terminal part of the linker peptide was constructed with the forward primer 5'-GCG TCT AGA GCT TCA GTC GTC AGC ATG GCT CGC-3' and the reverse primer 5'-CGC GGA TCC GCC ACC TCC CAG TTT TTC AAA GTT GAT TAT ACT AGC ATA CAA GCC GGT CAG-3' for OVA257–264 (SIINFEKL), 5'-CGC GGA TCC GCC ACC TCC GAG CCA CAC AAA AAA GTC ATA CAC AGC ATA CAA GCC GGT CAG-3' for tyrosinase-related protein 2 (TRP-2)181–188 (VYDFFVWL), or 5'-CGC GGA TCC GCC ACC TCC CGG CTG GGC TGT GTT ACA CTC AAA AGC ATA CAA GCC GGT CAG-3' for MUT1 (FEQNTAQP) (30). Cloning of a BamHI/XhoI fragment encoding mature human {beta}2m (h{beta}2m) with the C-terminal part of the linker peptide and the N-terminal part of the bridge was described (27). An analogous stretch containing the mature m{beta}2m was cloned by RT-PCR using the forward primer 5'-GGC GGA TCC GGA GGT GGT TCT GGT GGA GGT TCGATC CAG AAA ACC CCT CAA-3' and the reverse primer 5'-AAG ACC GTC TAC TGG GAT CGA GAC ATG CTG AGA TGG GAG CCC-3'. The template for m{beta}2m gene segments was mRNA from the MD45 T cell hybridoma (H-2k/d), and the gene product encodes Asp at the polymorphic position 85. The production of an XhoI/NotI fragment encoding the peptide bridge and the transmembrane and cytoplasmic portion of H-2Kb was described elsewhere (28). All PCR products were subcloned, and their DNA sequence was verified. The complete genes were assembled via a single-step insertion of the three corresponding fragments into the multiple cloning site of either vector.

Mice and cell lines

Eight- to 12-wk-old C57BL/6 (B6) mice were purchased from The Jackson Laboratory and bred at the Weizmann Institute of Science (WIS) facilities. Animals were maintained and treated according to the WIS animal facility and National Institutes of Health guidelines.

RMA is a Rauscher virus-transformed lymphoma cell line of B6 (H-2b) origin and RMA-S is an RMA TAP-deficient mutant (31). RMA-S:OVA is an RMA-S transfectant expressing OVA, prepared in our laboratory (E. Bar-Haim and L. Eisenbach, unpublished observations). Cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 50 µm 2-ME, and combined antibiotics. B3Z (32), an OVA257–264-specific, Kb-restricted CTL hybridoma, which expresses the NFAT-LacZ reporter gene, was a kind gift from Dr. N. Shastri (University of California, Berkeley, CA). F10.9 is a spontaneously metastasizing clone of the B16 melanoma (33). MO5, an OVA gene-transfected B16 (34), was a kind gift from Dr. K. Rock (University of Massachusetts Medical School, Worcester, MA). MO5 cells were maintained in DMEM supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 1% sodium pyruvate, 1% nonessential amino acids, combined antibiotics, and 500 µg/ml G-418 (Invitrogen Life Technologies).

Abs, proteins, and peptides

mAb against h{beta}2m (clone BM-63) was from Sigma-Aldrich. Polyclonal rabbit anti-h{beta}2m Abs were from DakoCytomation. The 25-D1.16 hybridoma, secreting a mAb specific to Kb-OVA257–264 (35), was a kind gift from Dr. R. Germain (National Institutes of Health, Bethesda, MD). mAb 20-8-4 is specific to H-2Kb, and 28-14-8 is specific to H-2Db. Abs were purified from hybridoma supernatants. Purified h{beta}2m was from Sigma-Aldrich. OVA257–264 and TRP-2181–188 were synthesized by Dr. M. Fridkin (WIS).

DNA transfection

RMA-S (0.8 ml) at 4 x 106 cells/ml were mixed in a 4-mm sterile electroporation cuvette (ECU-104; EquiBio) with 20 µg of linearized plasmid DNA. Transfection was performed with an Easyject Plus electroporation unit (EquiBio) at 250 V, 750 µF. Cells were resuspended in fresh medium and cultured for 24–48 h in 96-well plates before the addition of G418 to a final concentration of 1 mg/ml. Resistant clones were expanded in 24-well plates and screened by flow cytometry for expression of h{beta}2m or increase in expression of surface H-2Kb.

Flow cytometry

Cells (106) were grown for 24 h in serum-free medium (OptiMEM; Invitrogen Life Technologies), washed with FACS buffer (PBS, 5% FCS, and 0.05% sodium azide), and incubated for 30 min on ice with 100 µl of first (or control) Ab at 10 µg/ml. Cells were then washed and incubated on ice with 100 µl of 1/100 dilution of goat anti-mouse IgG (Fab-specific)-FITC-conjugated polyclonal Abs (Sigma-Aldrich) for 30 min, washed, resuspended in PBS, and analyzed with FACSCalibur (BD Biosciences). Mean fluorescence intensity was calculated using CellQuest software (BD Biosciences). Quantitative analysis of cell surface Ags was performed with QIFIKIT (DakoCytomation) according to the manufacturer’s instructions.

B3Z activation

Bulk LacZ assay was performed essentially as described (36) with some modifications. Stimulators (transfectants or RMA-S cells preloaded with OVA257–264) and responder B3Z hybridoma cells were washed three times with PBS, resuspended in nonselective medium at 5 x 105 cells/ml, and 50 µl of each was added to flat-bottom wells of a microtiter plate and coincubated overnight at 37°C. Cells were then resuspended and centrifuged at 1200 rpm for 5 min at room temperature. The supernatant was aspirated, and the cell pellets were lysed by the addition of 100 µl of Z buffer containing 100 mM 2-ME, 9 mM MgCl, 0.125% NP40, and 0.3 mM chromophenol red {beta}-galactoside (Fluka); incubated at 37°C in dark, and read at 570- and 630-nm reference wavelengths with a 96-well plate ELISA reader (Sunrise; Tecan) at different time points.

Immunization, cell cytotoxicity, and tumor-protection experiments

Groups of 12 8- to 12-wk-old female B6 mice were immunized three times i.p. with irradiated transfectants, peptide-loaded RMA-S, or control cells (2 x 106/0.5 ml, 5000 rad) at 7-day intervals. Peptide-loaded cells were prepared as follows: Cells were washed, resuspended at 5 x 106/ml in OptiMEM (Invitrogen Life Technologies), and incubated with 50 µg/ml synthetic peptide for 2 h at 26°C and then for 4 h at 37°C. On day 12 after the third immunization, two mice from each group were sacrificed, and their spleens were removed. Splenocytes were restimulated in vitro for 5 days in the presence of either OVA257–264 or TRP-2181–188 at 50 µg/ml. Viable lymphocytes were isolated on lympholyte-M gradient (Cedarlane Laboratories) and incubated at different E:T ratios with L-[35S]methionine-labeled target cells for 4 h. The percentage of specific lysis of triplicates was calculated as follows: (average experimental cpm – average spontaneous cpm)/(average maximum cpm – average spontaneous cpm) x 100. Maximal 35S release was obtained by lysis of target cells in 0.1 M NaOH. YAC-1 murine lymphoma cells were added to all cytotoxicity experiments as controls for NK-mediated cytolysis. For evaluation of tumor protection, on day 12 after the third immunization, mice were injected s.c. in the upper back with 1 x 105 MO5 tumor cells. Two perpendicular diameters of the tumor were measured with calipers. Mice were monitored daily and sacrificed when moribund. Survival was defined as the day when mice were sacrificed.

Tumor immunotherapy

Ten mice in each experimental group were inoculated s.c. in the upper back with 1 x 105 MO5 cells/mouse. Local tumor diameter was measured with calipers. Starting 8 days later, when the tumor reached 3–4 mm in diameter, mice were immunized i.p. four times at 7-day intervals with 2 x 106 irradiated transfectants or control cells preloaded with peptide at 50 µg/ml. Tumor diameter and survival were recorded.

Statistical analysis

Statistical differences in tumor sizes between groups of mice was determined by one-way ANOVA. Significance of survival plots was done with Kaplan-Meier survival platform. For both analyses, we used the JMP statistics software (SAS Institute).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Assembly of chimeric genes encoding antigenic peptides linked to membranal {beta}2m

In a recent report (28), we described the genetic engraftment of the transmembrane and cytoplasmic domains of H-2Kb as the anchor element of membranal {beta}2m. Although it has been argued that the MHC-I H chain is capable of transducing signals via the JAK/STAT pathway following MHC-I cross-linking, this capacity is independent of the transmembrane and cytoplasmic domains (37, 38). In our T cell-based experimental system, MHC-I ligation did not activate transfectants (Ref.27 and A. Margalit, S. Fishman, and G. Gross, unpublished observations). We thus refer to the H-2Kb portion as an inert anchor. Human {beta}2m possesses high affinity for most mouse MHC-I allelic products, including H-2Kb (39, 40), and can serve as a useful expression marker for gene-modified mouse cells. However, a possible effect of this disparity on immunogenicity could not be ruled out. To allow preliminary assessment of such an influence, we assembled and compared genetic constructs comprising both h{beta}2m and m{beta}2m, attached to the cell membrane via the H-2Kb appendage. As a tumor model, we chose MO5, which expresses both chicken OVA as a xenoantigen, providing the immunodominant H-2Kb-binding OVA257–264 peptide and TRP-2, a self-melanocyte differentiation Ag, harboring the poorly immunogenic peptide TRP-2181–188, which binds H-2Kb with low affinity. Sketches of the double-chimeric genes and the resulting MHC-I complexes, and designation of the four constructs examined in this study are presented in Fig. 1.



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  		FIGURE 1. dc{beta}2m: genetic scheme, resulting MHC-I product, and stable RMA-S transfectants. A, Major restriction sites are shown. pr, Promoter; lead, leader peptide; li, linker peptide; p, antigenic peptide; br, bridge. B, Anticipated configuration of the polypeptide product in the context of an MHC-I H ({alpha}) chain at the cell membrane. C, Table listing the four RMA-S clones explored in this study.

 
Expression of dc{beta}2m in RMA-S cells

RMA-S is an H-2b lymphoma cell line that does not express functional TAP. Due to the lack of high-affinity peptides normally transported from the cytosol, its MHC-I molecules are thermolabile, and their surface level at 37°C is markedly reduced. MHC-I level is restored at lower temperatures (26–28°C) or when high-affinity peptides are supplied in a TAP-independent manner. Binding of such peptides can be easily monitored by the concomitant increase in surface MHC-I level. In vivo, RMA-S cells loaded with such peptides are potent CTL inducers. RMA-S cells were transfected with the four dc{beta}2m-encoding plasmids, and G418-resistant clones were expanded and screened by FACS for expression of the introduced genes. Four clones were selected and expanded: Y314-7(mOVA), Y317-2(hOVA), Y316-8(mTRP), and Y318-10(hTRP). Comparative FACS analysis of Y317-2(hOVA), RMA, and parental RMA-S cells using Abs against H-2Kb, H-2Db, and H-2Kb-OVA257–264 complex was performed at 27 and 37°C (Fig. 2A). This analysis indicates that, at 37°C, dc{beta}2m elevated the level of not only the cognate MHC-I product (H-2Kb), but also of H-2Db. This phenotype was typical to all dc{beta}2m-expressing clones screened (data not shown). Staining of Y317-2(hOVA) with the H-2Kb-OVA257–264-specific Ab reveals that the covalently linked antigenic peptide in the dc{beta}2m polypeptide was indeed presented independently of the normal peptide processing and loading machinery. A quantitative analysis of surface MHC-I molecules in an independent experiment (Table I) shows that ~43% of surface H-2Kb molecules expressed by Y317-2(hOVA) were occupied with OVA257–264 at 37°C.



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  		FIGURE 2. Functional expression of dc{beta}2m. A, FACS analysis of OVA257–264/h{beta}2m/Kb by Y317-2(hOVA). The indicated cells were grown in serum-free medium for 24 h at 27°C (filled histogram) or 37°C (thick line). Cells were then incubated with mAbs to H-2Db (28-14-8), H-2Kb (20-8-4), Kb-OVA257–264 complex (25-D1.16) or no Ab (dotted line), and then with FITC-conjugated goat anti-mouse Fab Abs and analyzed by FACS. Fluorescence intensity is presented in logarithmic scale. B, Specific activation of the B3Z T cell hybridoma by Y317-2(hOVA). Transfectants Y317-2(hOVA), Y318-10(hTRP), and RMA-S cells with no peptide or preloaded with OVA257–264 (OVA) at the indicated concentrations, were incubated overnight at a 1:1 ratio with B3Z cells, a CTL hybridoma specific to the Kb-OVA257–264 complex, which expresses the NFAT-LacZ reporter gene. T cell activation was monitored by a {beta}-Gal enzymatic assay using chromophenol red {beta}-galactoside. Absorbance was monitored with an ELISA reader at 570 and 630 nm as a reference wavelength. Data from four independent experiments are shown.

 

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  		Table I. Absolute numbers of cell surface MHC-1 molecules expressed at 37°C on RMA-S cells in the absence or presence of OVA257–264 and on Y317-2(hOVA)

 
To test whether the genetically linked OVA257–264 can activate Ag-specific T cells, we incubated Y317-2(hOVA) with the B3Z T cell hybridoma and monitored LacZ enzymatic activity (Fig. 2B). Whereas RMA-S alone or Y318-10(hTRP) failed to activate B3Z, substantial activation was achieved following coincubation with Y317-2(hOVA), which was evident in numerous experiments. The magnitude of this activation was comparable to that evoked by RMA-S cells loaded with 0.1 ng/ml OVA257–264, although the data obtained with the complex-specific Ab (Table I) imply that peptide density on Y317-2(hOVA) is similar to the density on RMA-S in saturating peptide concentrations. These findings indicate that neither the C-terminal extension of OVA257–264 nor the space occupied by the Gly4Ser(Gly3Ser)2 linker peptide abolishes T cell recognition by the B3Z hybridoma. However, this design may impose some distortion on the particular epitope recognized by B3Z, which influences the magnitude of the hybridoma response but does not affect recognition by the complex-specific Ab.

Induction of peptide-specific CTLs in vivo

We next evaluated the ability of the RMA-S transfectants to elicit peptide-specific CTLs in vivo in comparison with other cell vaccines. Fig. 3 presents results from cell cytotoxicity assays performed after three immunizations. CTLs expanded from B6 mice immunized with Y314-7(mOVA) and Y317-2(hOVA) were more potent in killing MO5 cells than CTLs obtained following immunization with peptide-pulsed RMA-S cells (Fig. 3A). As expected, only a minimal CTL response was induced in mice receiving RMA-S:OVA. Similarly, Y316-8(mTRP) and Y318-10(hTRP) were far more potent than RMA-S cells loaded with TRP-2181–188 in inducing TRP-2181–188-specific CTLs, as monitored by cytolytic activity against TRP-2181–188-pulsed RMA-S cells (Fig. 3B). No response against RMA-S presenting TRP-2181–188 was induced in this experiment by RMA-S cells saturated with OVA257–264. Peptide specificity of the response was further addressed by using RMA-S cells charged with OVA257–264 as target cells of the same CTL populations presented in Fig. 3, B and C. No or only minimal lytic activity was now manifested by Y316-8(mTRP) and Y318-10(hTRP), respectively.



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  		FIGURE 3. In vivo induction of CTLs by RMA-S transfectants expressing dc{beta}2m constructs. The data are presented as percent specific lysis of MO5 (A), RMA-S cells charged with TRP-2181–188 (B), and RMA-S cells charged with OVA257–264 (C), both at 50 µg/ml. In each experiment, irradiated cells (dc{beta}2m RMA-S transfectants, RMA-S cells pulsed with 50 µg/ml peptide or RMA-S:OVA, which are RMA-S cells expressing chicken OVA; see upper inset) were inoculated three times i.p. at 7-day intervals. Twelve days after the last immunization, splenocytes were restimulated in vitro for 5 days with synthetic peptide (OVA257–264 or TRP-2181–188, respectively) and were then subjected to a cell cytotoxicity assay with L-[35S]methionine-labeled target cells at the indicated ratios. Data are representative of three independent experiments with similar results. The range of CTL activities was <5% of the mean of the triplicates in these experiments.

 
To address a possible influence of NK reactivity on our results, we included YAC-1 cells in our cell cytotoxicity assays. Although YAC-1 cells were effectively lysed in these assays (data not shown), no unspecific killing of RMA-S cells loaded with OVA257–264 (Fig. 3B) or TRP-2181–188 (C) was evident. Therefore, the observed differences in CTL induction are unlikely to result from faster in vivo clearing of peptide-loaded RMA-S cells (now displaying elevated MHC-I) by NK cells, compared with transfectants.

In these experiments, transfectants expressing peptide fused to h{beta}2m evoked CTL reactivity, which was comparable to cells expressing the same peptide fused to m{beta}2m, and this also was evident after only two immunizations (data not shown). These observations seem to rule out immunological bias that may have been imposed by the human component in Y317-2(hOVA) and Y318-10(hTRP). Notably, CTLs induced by cells presenting a covalently bound peptide are reactive against target cells presenting the native peptide, in accord with B3Z activation by Y317-2(hOVA).

Induction of protective antitumor immunity by transfectants

We then tested the ability of dc{beta}2m cell-based vaccines to provide protection against melanoma. To this end, we immunized B6 mice three times with Y317-2(hOVA) or Y318-10(hTRP) and with RMA-S cells preloaded with OVA257–264 or TRP-2181–188 peptide for comparison or with the 3LL Lewis lung carcinoma-associated MUT1 peptide as a negative control. Twelve days after the last inoculation, mice were challenged with 1 x 105 MO5 melanoma cells, and tumor size, along with animal survival, were recorded. As shown in Fig. 4A, expression of the TRP-2181–188-bearing construct by Y318-10(hTRP) cells had a significant protective effect, compared with TRP-2181–188-saturated RMA-S cells (p < 0.0001). Six of eight mice that received Y318-10(hTRP) remained tumor-free 7 wk after tumor challenge, compared with two of seven mice immunized with TRP-2181–188-loaded cells (p = 0.04) and only one of eight following immunization with MUT1-loaded cells (Fig. 4B). All mice (eight of eight) immunized with Y317-2(hOVA) remained tumor-free during the same period, compared with six of eight mice immunized with RMA-S cells saturated with OVA257–264. However, in light of the relative efficacy of OVA257–264-pulsed cells, no statistical significance could be derived for the latter set of data.



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  		FIGURE 4. Comparative analysis of tumor protection conferred by RMA-S transfectants and peptide-loaded RMA-S cells. Groups of 10 8- to 12-wk-old female B6 mice were immunized three times i.p. with the indicated irradiated cells. Twelve days after the last immunization, MO5 tumor cells were administered s.c. A, Inhibition of tumor growth. Local tumor dimensions were measured with calipers. The average of tumor diameters (in millimeters) in the course of 50 days is presented. The results are presented as mean + SEM. p value is shown for the group immunized with Y318-10(hTRP) against the group immunized with TRP-2181–188-loaded RMA-S. B, Survival of immunized mice. Mice from the same experiment were monitored daily and were sacrificed when moribund, which corresponded to a tumor diameter of ~20 mm. Fraction of surviving mice in each group is presented. p value is shown for the same groups as in A.

 
Immunotherapy of tumors

To evaluate immunotherapy of melanoma, we performed an experiment of tumor growth inhibition. B6 mice were challenged with 1 x 105 MO5 cells each. Starting 8 days later, mice were subjected to an immunization regimen with irradiated Y317-2(hOVA), parental RMA-S cells pulsed with OVA257–264, or with PBS only as control. As evident from Fig. 5A, tumor growth was significantly delayed in mice vaccinated with Y317-2(hOVA), compared with the peptide-loaded cells (p < 0.0001). This therapeutic effect also was evident from the survival graph (Fig. 5B). Of 10 mice vaccinated with Y317-2(hOVA), 8 were still alive 7 wk after tumor challenge, compared with 3 of 10 of mice vaccinated with RMA-S cells loaded with the peptide (p < 0.0001) and 0 of 10 of nonimmunized mice. In contrast, immunization with Y318-10(hTRP) and TRP-2181–188-loaded RMA-S cells under the same experimental conditions failed to yield any significant MO5 suppression effect (data not shown).



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  		FIGURE 5. Inhibition of tumor growth. MO5 tumor cells (1 x 105/mouse) were injected s.c. to female B6 mice (8–12 wk old). Eight days later, when tumor diameter reached 3–4 mm, mice were divided to groups of 10 and were immunized i.p. four times at 7-day intervals (days 8, 15, 22, and 29) with irradiated Y317-2(hOVA) or RMA-S cells loaded with 50 µg/ml OVA257–264 or with PBS only (nonimmunized). A, Tumor progression. Local tumor dimensions were measured with calipers. The average of tumor diameters (in millimeters) in the course of 50 days is presented. B, Survival of immunized mice. Mice from the same experiment were monitored daily and were sacrificed when moribund, which corresponded to a tumor diameter of ~20 mm. Fraction of surviving mice in each group is presented. Data are representative of two independent experiments with similar results. The results are presented as mean + SEM. Both A and B present p values calculated for the two groups of immunized mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we evaluated dc{beta}2m genetic constructs as a basis for cancer vaccines designed to maximize presentation of CTL epitopes by gene-modified APCs. The elucidation of discrete steps in the molecular pathway leading to MHC-I presentation has identified major bottlenecks in this process. Their combined effect mounts to an estimated 1:2000 ratio between cell surface MHC-I complexes comprising a particular peptide and the copy number of its polypeptide precursor (11). Engineering MHC-I components as protein vehicles for targeting N-terminally linked peptides to the ER aims at increasing this ratio. Aside from obviating the need for proteasomal processing of the Ag and transportation of peptides to the ER, this design maximizes peptide concentration at the site of complex assembly. There, it is likely to promote binding when a properly folded MHC-I H chain and {beta}2m are first brought into close proximity. Another important contribution to enhanced presentation can emanate from reformation of intact complexes at the cell surface, because all components remain available following peptide dissociation. Our previous work, which explored peptideless membranal {beta}2m in RMA-S cells (28), suggested that these polypeptides were abundant at the cell surface in an uncoupled form. As such, they could rapidly associate with free H chains through lateral diffusion and form intact complexes in the presence of MHC-I binding peptides. In analogy, MHC-I complexes can be formed at the transfectant surface membranes by association of dc{beta}2m with free H chains, which become transiently available as soon as both peptide and {beta}2m disengage (41, 42). Such de novo formation of MHC-I complexes constitutes another potential source for CTL ligands, which can further contribute to CTL induction.

The elevation in MHC-I level in Y317–2(hOVA) is observed not only for H-2Kb (which binds OVA257–264) but also for H-2Db (Fig. 2 and Table I). This increase is manifested both by a marked elevation at 37°C (>3.5-fold) and by a higher 37–27°C ratio, compared with the parental RMA-S cells (0.36 vs 0.21). It is conceivable that MHC-I trimers are formed, which comprise membranal {beta}2m, but not the genetically linked antigenic peptide. Similar conclusions also were drawn from our previous experiments with transfected T cells expressing peptide-{beta}2m constructs, which harbor the CD3 {zeta}-chain anchor (Ref.27 and A. Margalit, S. Fishman, D. Berko, and G. Gross, unpublished observations). These dc{beta}2m chains, which temporarily form MHC-I complexes independently of the linked peptide, thus serve as an additional reservoir for the formation of the anticipated CTL ligands.

The use of the short flexible peptide for linking the C terminus of the antigenic peptide to the N terminus of {beta}2m necessitates the opening of the normally closed end of the MHC-I peptide-binding pocket to allow C-terminal protrusion. The anticipated structural distortion is adjacent to the C-terminal anchor residue and, as such, is potentially detrimental both to proper positioning within the MHC-I binding groove and to subsequent T cell recognition. Nevertheless, longer peptides have been shown to bind to different MHC-I molecules through C-terminal extensions (43, 44, 45, 46, 47, 48, 49, 50, 51). Moreover, previous works studying antigenic peptides covalently linked to either MHC-I H chain (21, 22, 52) or {beta}2m (23, 24, 25, 26, 27, 53, 54) have collectively shown that the C-terminal protrusion imposed by the synthetic linker is tolerated both with respect to MHC-I binding and to T cell recognition when used with H-2Db, -Dd, -Kb, -Kd, -Kk, -Ld, and HLA-A2. Our present study provides further support to the cumulative evidence that the CTL response mounted against a covalently linked ligand is highly effective against target cells presenting the nominal peptide. However, our findings cannot rule out contribution of nominal peptide, which is cross-presented by host DCs following internalization of transfectant-derived dc{beta}2m polypeptides.

Peptide-based immunogens have short half-lives in vivo, and presentation of peptides preloaded onto APCs terminates once they detach from the MHC-I heterodimer. In contrast, genetic vaccines drive long-term expression of the immunogen. This point is of crucial importance in vivo, because the time elapsing between binding of synthetic peptides and engagement with CTL precursors in secondary lymphoid organs may well exceed the peptide life span at the MHC-I binding groove, especially for low- to medium-affinity peptides (55). The use of heteroclitic peptides partially overrides this shortcoming by endowing the antigenic peptide with longer MHC-I half-life (56, 57, 58, 59, 60). Still, in vivo superiority of the genetic dc{beta}2m modality over peptide-loaded APCs also is manifested with the high-affinity OVA257–264 peptide. This finding argues that sustained supply of the CTL epitope for effective CTL induction is advantageous even in the experimental system that we used, in which most irradiated APCs die within 3 days after inoculation (E. Tzehoval and L. Eisenbach, unpublished observations).

Interestingly, although cells expressing the low-affinity TRP-2181–188 peptide elicited potent CTLs and induced protective antimelanoma immunity (Figs. 3 and 4), they failed to suppress the growth of pre-established tumors (data not shown). Although we have not investigated this phenomenon further, we favor the following explanation. Cumulative findings ascribe a critical role for regulatory T cells (Tregs) in suppressing CTLs specific to self-, nonmutated TAAs. In particular, the low immunogenicity of the B16 melanoma has been largely attributed to CD4+CD25+ Treg activity (61, 62, 63), and depletion of this subpopulation enhanced B16 rejection. Importantly, reversal of tolerance correlated with a marked increase in the number of TRP-2180–188-specific CTLs and with autoimmune skin depigmentation, demonstrating the disruption of normal immunoregulatory mechanisms (62). In the immunotherapy setting, the initial administration of MO5 cells in a nonimmunostimulatory context may have enhanced the activity of such Tregs to a level that prevented later induction of TRP-2-specific CTLs by Y318-10(hTRP). In contrast, in the tumor protection experiments, RMA-S cells were inoculated first. RMA-S cells are known to provide potent costimulatory signals to T cells (64), and these also may exert Treg inhibitory effects. Hence, protective immunization with RMA-S-derived clones may have sufficed to curtail TRP-2 tolerance and give rise to Ag-specific CTLs and subsequent antitumor immunity. Although other explanations for the inability of Y-318(hTRP) to suppress MO5 cannot be ruled out, this finding reiterates the need to counteract Treg activity as a prerequisite for the elicitation of antitumor CTLs reactive against nonmutated self-Ags.

A number of studies have attempted to augment CTL responses by increasing the density of particular CTL ligands on APCs through genetically engineered MHC-I components harboring antigenic peptides of choice. Several of these examined soluble proteins, either in the form of MHC-I trimers (21, 22), in which the antigenic peptide was fused with the N terminus of an MHC-I H chain, or peptide-{beta}2m fusions (23, 24, 53, 54). Although the anticipated structural and antigenic features were indeed demonstrated, none of these reports presented data addressing the induction of peptide-specific CTLs in vivo. Expression of such chimeric proteins by gene-transfected or virally transduced cells also has been reported, and these works are more relevant to our approach. Indeed, a peptide-H-2Kd construct expressed by transfected L cells conferred tumor protection in vivo (21). However, the immunological mechanism underlying this protection remained unclear, because no induction of peptide-specific CTLs in vivo was demonstrated. One of the above reports (24) also described the generation of human cell lines encoding an HLA-A2-binding influenza matrix epitope linked to the N terminus of h{beta}2m but provided no in vivo data. The only in vivo data described by two recent reports that examined peptide-{beta}2m-H-chain MHC-I single-chain trimers (25, 26) concern the mounting of an Ab response following DNA immunization. Taken together, the work presented here is the first to provide in vivo evidence that cells expressing a peptide-{beta}2m fusion induce tumor-specific CTLs, confer tumor protection, and inhibit tumor growth, which are the hallmarks of experimental antitumor CTL immunity.

In contrast to the use of an MHC-I H chain as the genetic scaffold for the antigenic peptide (e.g., Refs.21 and 22), the monomorphic nature of {beta}2m renders its membrane-anchored derivative an essentially universal vaccine platform: a single expression cassette should be compatible with all possible CTL ligands. Furthermore, the use of membranal {beta}2m offers a wide range of applications in cancer immunotherapy, such as the use of peptideless membranal {beta}2m, which improved the tumor-suppressing activity of peptide-loaded RMA-S cells in vivo (28).


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict 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 study was supported by a project grant from the Israel Cancer Research Fund and the Chief Scientist of the Ministry of Industry, Trade, and Labor (Israel). Back

2 A.M. and H.M.S. contributed equally to this study. Back

3 Current address: Department of Microbiology and Immunology, Faculty of Health Sciences and Cancer Research Center, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel. Back

4 Current address: Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel. Back

5 Address correspondence and reprint requests to Dr. Gideon Gross, MIGAL-Galilee Technology Center, P.O. Box 831, Kiryat Shmona 11016, Israel. E-mail address: gidi{at}migal.org.il Back

6 Abbreviations used in this paper: TAA, tumor-associated Ag; MHC-I, MHC class I; {beta}2m, {beta}2-microglobulin; DC, dendritic cell; ER, endoplasmic reticulum; dc, double chimeric; m, mouse; h, human; TRP-2, tyrosinase-related protein 2; Treg, regulatory T cell. Back

Received for publication June 22, 2005. Accepted for publication October 11, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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