Purpose: A prerequisite for the development of vaccination strategies is the identification and characterization of relevant tumor-associated antigen. Using microarray and reverse transcription-PCR analysis, we found matrix metalloproteinase (MMP)-7 to be extensively up-regulated in renal cell carcinomas and expressed in a broad variety of malignant cells. MMP-7 can promote cancer invasion and angiogenesis by proteolytic cleavage of extracellular matrix and basement membrane proteins, thus making it a promising target in the context of immunotherapies.
Experimental Design: To analyze the possible use of MMP-7 as a tumor-associated antigen, specific CTLs were induced using monocyte-derived dendritic cells electroporated with MMP-7-mRNA. In addition, to better characterize the fine specificity of these CTLs, MMP-7 MHC class I ligands were isolated and characterized in renal cell carcinoma tissue, which overexpressed MMP-7, by mass spectrometry–based peptide sequencing. Using this approach, we identified a novel HLA-A3–binding antigenic MMP-7 peptide. CTLs generated from healthy donors by in vitro priming with dendritic cells, pulsed with the novel peptide, were used as effectors in 51Cr-release assays.
Results: The induced CTLs elicited an antigen-specific and HLA-restricted cytolytic activity against tumor cells endogenously expressing the MMP-7 protein. Furthermore, we were able to induce MMP-7–specific CTLs using peripheral blood mononuclear cells from a patient with acute lymphoblastic leukemia capable of recognizing the autologous leukemic blasts while sparing nonmalignant cells.
Conclusions: Our study describes the identification of a novel broadly expressed T-cell epitope derived from the MMP-7 protein that represents an interesting candidate to be applied in immunotherapies of human malignancies targeting both tumor cells and neovascularization.
Identification and targeting of antigens involved in malignant transformation, survival advantage of tumor cells, or their metastatic spread might represent a promising approach in the development of immunotherapeutic strategies to treat malignant diseases.
Using microarray analysis of primary renal cell carcinoma (RCC) tissues, we recently found that matrix metalloproteinase (MMP)-7 is extensively up-regulated in malignant samples (1). MMPs are a family of structural-related zinc-dependent endopeptidases that degrade macromolecules of the extracellular matrix. Degradation of connective tissue is involved in many physiologic processes, such as embryogenesis, postpartum uterine involution, bone turnover, tissue repair, and angiogenesis as well as pathologic processes such as arthritis, decubitus ulcer, and tumor invasion. The gene family of the human MMPs comprises at least 19 members. They are divided into four classes, either in respect of their domain structure and preferred substrate (e.g., collagenases and gelatinases) or due to their localization such as the class of the membrane-bound MMPs (membrane type, MT1; ref. 2). With the exception of stromelysin 3 (MMP-3) all free MMPs are secreted as inactive proenzymes and are activated by enzymatic cleavage of the propeptide domain (3, 4). After their secretion and activation, they are inhibited by the tissue inhibitors of metalloproteinases (5, 6). The balance between the amount of activated MMP and tissue inhibitors of metalloproteinases determines the effective MMP activity (7).
Matrilysin (MMP-7) was discovered in rats in 1980 by Sellers and Woessner (8), and the human homologous gene was cloned in 1988 (9). Unlike most MMPs, it is constitutively expressed by many epithelial cell types, often ductal epithelium of adult exocrine glands in skin, salivary glands, pancreas, liver, and breast, and by glandular epithelium of the intestine and reproductive organs (10, 11). It is also found in the lumenal surface of dysplastic glands in the early-stage human colorectal tumors.
MMP-7 has a wide substrate specificity and can promote cancer invasion by proteolytic cleavage of extracellular matrix and basement membrane proteins, such as fibronectin, collagen type IV, laminin, elastin, entactin, osteopontin, and cartilage proteoglycan aggregates. Furthermore, this enzyme activates other MMPs, such as proMMP-2 and proMMP-9, to facilitate tumor invasion and seems to mediate the proteolytic processing of tumor necrosis factor precursor and urokinase plasminogen activator (12–15). MMP-7 knockout mice have reduced intestinal tumorigenesis (16). In line with this finding, it was reported that MMP-7 expression in humans is correlated with invasiveness of cancer tissues of the esophagus, stomach, colon, liver, and pancreas (17–22). Furthermore, several studies showed that MMP-7 is overexpressed in a broad variety of malignancies making this antigen an interesting candidate for immunotherapeutic approaches (23).
Angiogenesis is a critical step in cancer progression to enlarge and invade surrounding tissues (24, 25). Previous studies showed that several MMPs are expressed in vascular endothelial cells adjacent to tumor cells, suggesting their involvement in these processes by forming openings within the extracellular matrix through which newly generated capillaries can intrude and extend. MMP-7 has been shown to accelerate the proliferation of human umbilical vein endothelial cells in a dose-dependent manner in vitro (26). In another study, angiogenesis at the site at which human colon cancer cells were implanted in a mice model could be inhibited by a MMP-7–specific antisense oligonucleotide (27). Taken together, these findings suggest that this enzyme directly promotes angiogenesis, at least in part, through proliferative action on vascular endothelial cells. Thus, targeting MMP-7 may affect both tumor cells and tumor angiogenesis. Consequently, we initially examined the possible function of this molecule as a novel tumor-associated antigen using antigen-specific CTLs induced in vitro by monocyte-derived dendritic cells (DC) that have been electroporated with synthetic mRNA coding for MMP-7 as antigen-presenting cells (APC). We show that these CTLs elicited an antigen-specific and HLA class I–restricted cytolytic activity against tumor cells endogenously expressing the MMP-7 protein. Thereupon, we isolated and characterized MMP-7 MHC class I ligands in RCC tissue that overexpressed MMP-7, by mass spectrometry–based peptide sequencing (1, 28–31). Using these peptides, we were able to induce CTLs in vitro that elicited an antigen-specific and HLA-A3–restricted cytolytic activity against tumor cells expressing MMP-7. In a last series of experiments, we show that MMP-7–specific CTLs induced by DCs transfected with MMP-7–in vitro transcript (IVT) efficiently lysed autologous blasts from acute lymphoblastic leukemia (ALL) patients.
In conclusion, our study describes the identification of a novel broadly expressed T-cell epitope derived from the MMP-7 protein that represents an interesting candidate to be applied in immunotherapies of human malignancies capable of targeting neovascularization and malignant cells in developing tumors.
Materials and Methods
Tumor cell lines. All cells were cultured in RP10 medium (RPMI 1640 with glutamax I, supplemented with 10% inactivated FCS and antibiotics; Invitrogen). The cell lines used were A498, MZ1774, MZ1257 (RCC cell lines, HLA-A2+, kindly provided by Prof. A. Knuth, UniversitätsSpital Zürich, Zürich, Switzerland), CAKI-2 (RCC, HLA-A1+), U266 (multiple myeloma, HLA-A2+), SKOV-3 (ovarian cell line, HLA-A3+, kindly provided by O. J. Finn, University of Pittsburgh. Pittsburgh, PA), CROFT (EBV-immortalized B-cell line, HLA-A2+; kindly provided by O. J. Finn), THP-1 (human acute monocytic leukemia), NT-2 (NTERA-2, pleuripotent, embryonal carcinoma cells), KASUMI-1 (acute myeloid leukemia), WERI-RB-1 (human retinoblastoma), HL-60 (human acute myeloid leukemia), and K-562 (human chronic myeloid leukemia, HLA-A2+).
Generation of DCs from adherent peripheral blood mononuclear cells. DCs were generated from peripheral blood monocytes as described previously (32, 33). In brief, peripheral blood mononuclear cells (PBMC) were isolated by Ficoll/Paque (Biochrom) density gradient centrifugation of blood obtained from buffy coats of healthy volunteers from the blood bank of the University of Tübingen. Cells were seeded (1 × 108 cells/10 mL per flask) into 275-mL tissue culture flask (Becton Dickinson) in serum-free X-VIVO 20 medium (BioWhittaker). After 1.5 h of incubation at 37°C/5% CO2, nonadherent cells were removed and cryopreserved at −80°C to be used later for cell isolation or restimulations. Human recombinant granulocyte macrophage-colony stimulating factor (100 ng/mL; Leukine Liquid Sargramostim; Berlex) and interleukin-4 (20 ng/mL; R&D Systems) were added every second day starting at the first day of culture to generate immature DCs. Maturation was induced at day 6 of culture by adding 2.0 μg/mL R848 (TLR7/8L; InvivoGen) for 24 h.
Synthetic peptides.MMP-7 A*03: SLFPNSPKWTSK, RGS5 A*03: GLASFKSFLK (1), were synthesized in an automated peptide synthesizer EPS221 (Abimed) after the F-moc/tBu strategy and analyzed by high performance liquid chromatography (Varian Star; Zinsser Analytics) and MALDI-TOF mass spectrometry (future; GSG).
Gene expression analysis by high-density oligonucleotide microarrays. Frozen fragments of tumors RCC0044, 0068, 0070, 0073, 0075, 0098, and 0103 were homogenized with mortar and pestle under liquid nitrogen. Total RNA was prepared from these samples using TRIzol (Invitrogen) according to the manufacturer's protocol, followed by a cleanup with RNeasy (QIAGEN). Quality and quantity were assessed on an Agilent 2100 Bioanalyzer (Agilent) using the RNA 6000 Pico LabChip kit (Agilent). Gene expression analysis of RNA samples from RCC0044, 0068, 0070, 0073, 0075, 0098, and 0103 were done by Affymetrix Human Genome U133A oligonucleotide microarrays (Affymetrix). For all other samples, HG-U133 Plus 2.0 were used. The same normal kidney sample was hybridized to both array types to achieve comparability (data not shown). All steps were carried out according to the Affymetrix manual.4
Briefly, double-stranded cDNA was synthesized from 5 to 8 μg of total RNA, using SuperScript RTII (Invitrogen) and the oligo-dT-T7 primer (MWG Biotech) as described in the manual. In vitro transcription was done with the BioArray High Yield RNA Transcript Labeling kit (ENZO Diagnostics, Inc.) for the U133A arrays or with the GeneChip IVT Labeling kit (Affymetrix) for the U133 Plus 2.0 arrays, followed by cRNA fragmentation, hybridization, and staining with streptavidin-phycoerythrin and biotinylated antistreptavidin antibody (Molecular Probes). Images were scanned with the Agilent 2500A GeneArray Scanner (U133A) or the Affymetrix GeneChip Scanner 3000 (U133 Plus 2.0), and data were analyzed with the MAS 5.0 (U133A) or GCOS (U133 Plus 2.0) software (Affymetrix), using default settings for all variables. Pairwise comparisons were calculated using the respective normal kidney array as baseline. For normalization, 100 housekeeping genes provided by Affymetrix were used.5
RNA isolation for reverse transcriptipn-PCR. Total RNA was isolated from tumor cell Iysates using RNeasy Mini anion-exchange spin columns (QIAGEN) according to the protocol for isolation of total RNA from animal cells provided by the manufacturer. Quantity and purity of RNA was determined by UV spectrophotometry. RNA samples were routinely checked by formaldehyde/agarose gel electrophoresis for integrity and stored at −80°C in small aliquots.
Reverse transcription-PCR. Up to 5.0 μg total RNA was subjected to a 20-μL cDNA synthesis reaction (Transcriptor First Strand cDNA Synthesis kit; Roche) using random primers. cDNA (1.0 μL) was used in a 50.0-μL PCR amplification reaction. To control the integrity of the RNA and the efficiency of the cDNA synthesis, 1.0 μL cDNA was amplified by an intron-spanning primer pair for the β2-microglobulin gene. For the MMP-7 and the β2-microglobulin cDNA, the PCR temperature profiles were as follows: 5 min pretreatment at 94°C and 25 (β2-microglobulin) or 32 (MMP-7) cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 60 s with a final extension at 72°C for 7 min. Primer sequences were deduced from published cDNA sequences. β2-microglobulin, 5′-GGGTTTCATCCATCCGACAT-3′ and 5′-GATGCTGCTTACATGTCTCGA-3′; MMP-7, 5′-GAGTGCCAGATGTTGCAGAA-3′ and 5′-AAATGCAGGGGGATCTCTTT-3′. Reverse transcription-PCR reactions (10.0 μL) were electrophoresed through a 3.0% agarose gel and stained with ethidium bromide for visualization under UV light.
Generation of MMP-7– and enhanced green fluorescent protein–in vitro transcripts. A 1.1-kb MMP-7 cDNA fragment was excised by EcoRI restriction enzyme digestion from plasmid pLXSN-MAT.WT (generously provided by L. M. Matrisian, Vanderbilt University, Nashville, USA) and subcloned into pcDNA3.1 (Invitrogen), which allowed in vitro transcription under the control of a T7 promoter. Plasmid, harboring the MMP-7 insert in the correct orientation, was linearized behind the polyadenylate tail by BamHI restriction enzyme digestion and in vitro transcribed using the T7 mMESSAGE mMACHINE kit (Ambion) according to the protocol provided by the manufacturer. Purification of IVTs was done with RNeasy Mini anion-exchange spin columns (QIAGEN) according to the RNA cleanup protocol provided by the manufacturer. Enhanced green fluorescent protein (EGFP)–IVT was generated as described previously (34). Quantity and purity of IVTs were determined by UV spectrophotometry. The IVTs were routinely checked by formaldehyde/agarose gel electrophoresis for size and integrity and stored at −80°C in small aliquots.
RNA electroporation of DCs. Electroporation of DCs with RNA was done as described previously (35–37). Briefly, on day 7 of culture, mature DCs were harvested, washed twice with X-VIVO 20 medium, and resuspended to a final concentration of 1 × 107 cells/mL. Subsequently, 200 μL of the cell suspension were mixed with 10 μg of RNA and electroporated in a 4-mm cuvette using an Easyject Plus unit (EquiBio/Peqlab). The physical variables were as follows: voltage of 300 V, capacitance of 150 μF, and resistance of 1540 Ω. After electroporation, cells were immediately transferred into RP10 medium supplemented with interleukin-4, granulocyte macrophage colony-stimulating factor, and tumor necrosis factor-α and returned to the incubator.
CTL induction using DCs transfected with RNA. DCs were electroporated with different sources of RNA as described above. After transfection, DCs were incubated for 24 h in serum-free X-VIVO 20 (BioWhittaker) medium supplemented with granulocyte macrophage-colony stimulating factor, interleukin-4, and tumor necrosis factor-α. For T-cell induction, 5.0 × 105 electroporated DCs were incubated with 3.0 × 106 autologous PBMCs in X-VIVO 20 (BioWhittaker). After 7 d of culture, cells were restimulated with RNA-electroporated DCs, and 2.0 ng/mL interleukin 2 (R&D Systems) was added on day 1, 3, and 5. The cytolytic activity of induced CTLs was analyzed on day 5 after the last restimulation in a standard 51Cr-release assay.
Induction of MMP-7–specific CTLs using peptide-pulsed DCs. For CTL induction, 5 × 105 DCs were pulsed with 50 μg/mL synthetic peptide for 2 h, washed, and incubated with 3.0 × 106 autologous PBMCs in RP10 medium. After 7 d of culture, cells were restimulated with autologous peptide-pulsed PBMCs, and 1.0 ng/mL human recombinant interleukin-2 (Genzyme) was added on days 1, 3, and 5. The cytolytic activity of induced CTL was analyzed on day 5 after the last restimulation in a standard 51Cr-labeled release assay.
Standard51Cr-release assays (CTL assays). CTL assays were done as described previously (38). In brief, target cells were transfected with RNA or pulsed with 50.0 μg/mL synthetic peptide for 2 h and labeled with 51Cr sodium chromate in X-VIVO 20 medium for 1 h at 37°C. Target cells (1.0 × 104) were transferred to a well of a round-bottomed 96-well plate. Varying numbers of CTLs were added to a final volume of 200.0 μL and incubated for 4 h at 37°C. At the end of the assay, supernatants (50.0 μL per well) were harvested and counted in a β-plate counter. The percentage of specific lysis was calculated as: 100 × (experimental release − spontaneous release)/(maximal release − spontaneous release). Spontaneous and maximal releases were determined in the presence of either X-VIVO 20 medium or 2% Triton X-100, respectively. Inhibition of HLA class I molecules was achieved by incubating target cells for 1 h before the assay with the monoclonal antibody (mAb) W6/32 (20.0 μg/mL) directed against HLA class I molecules. Antigen specificity of cell lysis was further determined in a cold target inhibition assay by analyzing the capacity of unlabeled PBMCs pulsed with MMP-7 peptide to block lysis of tumor cell lines.
IFN-γ ELISPOT assay. MMP-7–specific CTLs generated in vitro using autologous DCs pulsed with the MMP-7 peptide (2 × 105 cells per well) were incubated at a concentration of 2 × 105 cells per well in an anti-human IFN-γ antibody-coated (mAb 1-D1K; 10 μg/mL; Mabtech AB) 96-well plate with autologous PBMCs pulsed for 1 h with the MMP-7 or RGS5 peptide as a control. For the detection of spots, a biotin-labeled antihuman IFN-γ antibody (mAb 7-B6-1-Biotin; 2 μg/mL; Mabtech AB) was used. Spots were counted after 40 to 44 h of incubation using an automated ELISPOT reader (IMMUNOSPOT ANALYZER; CTL Analyzers LLC).
Expression analysis of MMP-7. Recently, we found MMP-7 to be overexpressed in RCC (1). To assess the possible use of MMP-7 as a target antigen for the development of vaccination therapies, reverse transcription-PCR analysis was done on human primary ALL cells and established tumor cell lines. As shown in Fig. 1, MMP-7 expression was found in CAKI-2, MZ1257, SKOV-3, CROFT, NT-2 (faint), A498, and HL-60 tumor cell lines as well as in blood samples of patients with ALL.
Induction of MMP-7–specific CTLs using DCs electroporated with MMP-7-IVT. In the first series of experiments, we sought to analyze the feasibility to induce MMP-7–specific CTLs. Therefore, monocyte-derived DCs were generated from the PBMCs of a healthy HLA-A-A3+ donor, electroporated with an excess of pure full-length MMP-7-IVT that coded for the entire MMP-7-protein and used as APCs for the in vitro induction of antigen-specific CTLs. DCs electroporated with the respective MMP-7-IVT were used as target cells in the standard 51Cr-release assays. As shown in Fig. 2A, these CTLs did lyse cells electroporated with MMP-7-IVT, whereas DCs transfected with irrelevant EGFP-IVT were spared.
To further confirm and extend our findings, we included the MMP-7– expressing HLA-A3+ tumor cell line SKOV-3 as target in a standard 51Cr-release assays using CTLs as above that were induced from an HLA-A3+ donor. As shown in Fig. 2B, the malignant cells were efficiently lysed by the CTLs. In contrast, the in vitro induced T cells did not recognize the HLA-A2+ cell line A498 that expresses MMP-7. K-562 cells were also spared, indicating that the cytotoxic activity was not natural killer cell mediated. Furthermore, the specific lysis of the target cells could be blocked using a mAb directed against HLA class I molecules demonstrating that the elicited T-cell responses were HLA class I restricted. Consequently, the antibody directed against HLA class II molecules did not inhibit the lysis of the tumor cells (Fig. 2B).
These data indicate that DCs electroporated with an excess of pure full-length MMP-7-IVT can result in the presentation of MMP-7–derived peptides on the cell surface that can induce MMP-7–specific CTLs that recognize MMP-7–positive target cells in an antigen-specific manner in vitro.
Induction of MMP-7–specific CTLs using peptide-pulsed DCs. In the next set of experiments, we wanted to identify HLA class I–presented MMP-7 peptides that are recognized by our CTL lines. To accomplish this, peptides were isolated from surgically removed RCC specimens that overexpressed MMP-7 according to standard protocols (39) using the HLA class I–specific antibody W6/32. By applying mass spectrometry–based peptide sequencing of the HLA ligands, a HLA-A3–binding peptide derived from the novel antigen MMP-7 (HLA-A*03 ligand SLFPNSPKWTSK) was identified (1).
To analyze the immunogenicity of the newly identified MMP-7–derived peptide with respect to its ability to induce T-cell responses, we induced MMP-7–specific CTLs in vitro. DCs derived from adherent PBMCs of HLA-A3+ healthy donors were pulsed with the HLA-A3–binding antigenic peptide and used as APCs. The cytotoxicity of the induced CTLs was assessed in standard 51Cr-release assays. As shown in Fig. 3A, the CTL line obtained after several weekly restimulations showed antigen-specific killing: T cells only recognized autologous DCs loaded with the cognate MMP-7-A3 peptide, whereas they did not lyse target cells pulsed with an irrelevant HLA-A3–binding RGS5 peptide, confirming the specificity of the cytolytic activity. Additionally, we included autologous DCs electroporated with MMP-7-IVT as targets in the cytotoxicity assay. As shown in Fig. 3A, these cells were efficiently lysed, whereas DCs electroporated with irrelevant EGFP-IVT were spared.
These findings indicate that DCs loaded with MMP-7–derived peptides can induce specific CTLs that recognize cells presenting MMP-7 epitopes in an antigen-specific and HLA-A–restricted manner. Furthermore, DCs electroporated with an excess of pure full-length MMP-7-IVT can result in the presentation of the newly identified MMP-7–derived peptide on the cell surface, thus confirming the processing and presentation of the HLA-A3–binding peptide.
In the next set of experiments, we evaluated the ability of the in vitro induced MMP-7–specific CTLs generated with HLA-A3–binding peptide to lyse tumor cells endogenously expressing MMP-7. The MMP-7– and HLA-A3–expressing cell line SKOV-3 was used as target in a standard 51Cr-release assay. The MMP-7–specific CTLs were able to efficiently lyse malignant cells expressing both HLA-A3 and MMP-7 (Fig. 3B). In contrast, we detected no lysis of the HLA-A2+ and MMP-7+ cell line A498, demonstrating that the CTLs were HLA-A3 restricted. The in vitro induced CTLs did also not recognize K-562 cells, indicating that the cytotoxic activity was not natural killer cell mediated.
To further confirm the antigen specificity, we did cold target inhibition assays. PBMCs loaded with the HLA-A3–binding MMP-7 peptide but not with the HLA-A3–binding RGS5 peptide could inhibit the recognition of HLA-matched SKOV-3 tumor cells, thus confirming that the in vitro induced CTLs are specific for the respective MMP-7 epitope (Fig. 3C). Furthermore, the specific lysis of the target cells could be blocked using a mAb directed against HLA class I molecules, indicating that the elicited T-cell responses were HLA class I restricted (Fig. 3C).
These data indicate that DCs pulsed with MMP-7–derived peptides can induce MMP-7–specific class I–restricted CTLs that recognize tumor cells endogenously expressing MMP-7 in an antigen-specific and HLA-A–restricted manner in vitro.
MMP-7–specific CTLs induced by DCs electroporated with MMP-7-IVT can lyse target cells pulsed with MMP-7 peptide. To further prove the presentation of the identified peptide, we next wanted to test whether MMP-7–specific CTLs induced by DCs electroporated with pure full-length MMP-7-IVT are capable of recognizing target cells pulsed with the newly identified MMP-7 peptides. As shown in Fig. 4, the induced CTLs did efficiently lyse DCs loaded with the MMP-7 A3 peptide, whereas cells pulsed with the irrelevant RGS5-A3 peptide as a control were spared. Additionally, the induced CTLs did lyse cells electroporated with MMP-7-IVT, whereas DCs transfected with irrelevant EGFP-IVT were spared.
Induction of MMP-7–specific CTLs from a patient with RCC. Using IFN-γ ELISPOT Assays, we found in 2 of 5 analyzed HLA-A3–positive patients with metastatic RCC MMP-7 peptide–specific T cells in peripheral blood with a frequency of 25.5 in 5 × 106 PBMCs and 39.5 in 106 PBMCs, respectively.
In our next experiments, we wanted to test the feasibility to induce MMP-7–specific CTLs in patients with RCC. Therefore, we generated monocyte-derived DCs from an HLA-A3+ RCC patient with metastatic disease and used them as APCs for CTL induction after pulsing with the HLA-A3–binding MMP-7 peptide. As presented in Fig. 5A, the induced CTLs did efficiently lyse the HLA-A3+ SKOV-3 tumor cell line. The specific lysis of these target cells could be blocked using a mAb directed against HLA class I molecules. As a control, we additionally included the HLA-A2+ tumor cell line A498 and the HLA-A1+ cell line CAKI-2. These cell lines were spared by the CTLs, confirming their HLA-A3+ restriction.
In addition, as shown in Fig. 5B, autologous DCs loaded with MMP-7 peptide or MMP-7-IVT were efficiently lysed by the induced CTLs, whereas DCs pulsed with irrelevant RGS5 peptide or EGFP-IVT were spared.
Taken together, these experiments indicate the feasibility to induce MMP-7–specific CTLs from RCC patients that efficiently recognize and lyse tumor cells endogenously expressing MMP-7.
MMP-7–specific CTLs induced by DCs electroporated with MMP-7-IVT can lyse autologous ALL-blasts. In the last series of experiments, we analyzed, whether our previous findings could be applied in an autologous setting. To achieve this, we generated monocyte-derived DCs from an HLA-A2+/-A3+ ALL patient in complete remission after chemotherapy and used them as APCs for CTL induction after electroporation with full-length MMP-7-IVT. As target cells, we included autologous DCs loaded with the respective peptide or transfected with MMP-7-IVT. As shown in Fig. 6A, these cells were efficiently lysed, whereas cells pulsed with an irrelevant RGS5 peptide or electroporated with EGFP-IVT were spared.
Additionally, we included the autologous ALL blasts frozen at the time of diagnosis and used them as targets in standard 51Cr-release assays. These cells were efficiently lysed by the CTLs specific for MMP-7, confirming the expression analysis by reverse transcription-PCR. Importantly, the normal autologous purified CD3+ T and CD19+ B cells (i.e., the normal lymphoid counterparts of the ALL blasts) were not recognized by the CTLs (Fig. 6B).
During the last decade, great efforts have been made to develop immunotherapeutic approaches for the treatment of malignant diseases as an additional strategy to chemotherapy and radiotherapy. A prerequisite for this purpose is the understanding of the molecular pathways required for induction and maintenance of the immunosurveillance and immunoresponse and the development of methodologies to generate tumor-specific antigen and/or tumor-associated antigen–specific CTLs or mAbs as immunologic therapeutic agents. According to this, the basic requirement for these approaches is the availability of well-defined and characterized tumor-specific antigens and/or tumor-associated antigens as target structures that allow the specific attack of malignant cells. In recent years, a large number of tumor-specific antigens and tumor-associated antigens have been identified, which can be recognized by T cells (40).
DCs are recognized as the most powerful professional APCs, with the ability to initiate primary immune responses in vivo. Several procedures to generate large number of DCs as adjuvant from circulating precursors, including peripheral blood monocytes and CD34+ progenitor cells, have been developed for clinical use. Hence, numerous attempts to optimize delivery of tumor antigens to DCs, as well as routes and schedules of administration to cancer patients, are currently being evaluated in clinical trials. Some of them showed promising results with the induction of tumor regression in a proportion of patients (41, 42).
The putative metalloproteinase I (PUMP1) gene was identified through studies of collagenase-related connective tissue–degrading metalloproteinases produced by human tumors. It was later called matrilysin or MMP-7. MMPs are enzymes implicated in normal and pathologic tissue–remodeling processes. MMP-7 was shown to be involved in the invasiveness and progression of cancer cells. Generalized, MMP-7 is expressed in many malignancies, thus representing an interesting target antigen for immunotherapeutic approaches. The high-density oligonucleotide microarray analysis revealed MMP-7 to be overexpressed in 4 of 11 RCC tumor samples analyzed (1), and we found MMP-7 expression in a broad variety of tumor cells, including renal and ovarian cell carcinomas, B-cell lines, embryonal carcinoma, acute myeloid leukemia, and primary ALL blasts.
Interestingly, MMPs can be produced by endothelial cells (43, 44). Endothelial synthesis of MMPs seems to have opposite effects on tumor angiogenesis, on the one hand, facilitating extracellular matrix degradation and new blood vessel formation (45) and, on the other hand, blocking angiogenesis by producing inhibitors of endothelial cell growth, including angiostatin (46). Taken together, targeting this molecule in tumors should not only affect the malignant cells but also inhibit neoangiogensis that accompanies tumor progression.
To generate MMP-7–specific CTLs, we first used PBMCs from HLA-A3+ healthy donors in an in vitro immunization protocol. CTL induction was carried out using monocyte-derived DCs that were electroporated with an excess of pure full-length MMP-7-IVT. The in vitro generated CTLs efficiently lysed DCs transfected with cognate antigenic RNA in 51Cr-release assays, demonstrating the feasibility of these approach and immunogenicity of MMP-7–derived peptides processed by the DCs. To determine the fine specificity of our HLA-A3–restricted CTL line, peptides were isolated and analyzed by mass spectrometry from primary tumor samples of patients with RCC overexpressing MMP-7. Using this approach, a HLA-A3–restricted peptide derived from the novel antigen MMP-7 was identified. This peptide was analyzed as a means to induce T-cell responses that mediate tumor cell lysis. The in vitro induced MMP-7-specific CTLs using DCs electroporated with IVT-RNA or MMP-7 peptide were not only able to lyse target cells pulsed with the antigenic peptide but also recognized tumor cells endogenously expressing the MMP-7 protein in an antigen-specific and HLA-restricted manner. The specificity of the lytic activity was confirmed by the addition of a mAb blocking the HLA molecules or by performing cold target inhibition assays.
Finally, we tested the ability of the identified MMP-7 peptides to elicit CTL responses in patients with malignant diseases. Therefore, CTL lines were generated from the PBMCs of a patient with metastatic RCC or ALL in complete remission. These MMP-7–specific CTLs were used as effectors against autologous DCs, HLA-matched tumor cells, or the autologous malignant cells frozen at the time of diagnosis. The in vitro induced CTLs efficiently lysed the autologous leukemic cells in a MHC-restricted and antigen-specific manner but spared normal B and T cells from this patient. These results showed that MMP-7–specific CTLs can be generated in patients with malignant diseases able to recognize primary autologous tumor cells.
In conclusion, our study describes the identification of novel broadly expressed tumor-associated antigen and a T-cell epitope derived from the MMP-7 protein that represents interesting candidate to be applied in immunotherapies of human malignancies.
Disclosure of Potential Conflicts of Interest
P. Brossart and H.-G. Rammensee have an ownership interest with Immatics Biotechnologies (Tübingen, Germany).
Grant support: Deutsche Forschungsgemeinschaft (SFB 685).
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.
Note: Y. Yokoyama and F. Grünebach contributed equally to this work.
We thank Regina Heselmaier, Bruni Schuster, and Sylvia Stephan for excellent technical assistance.