ADAM metallopeptidase domain 17 (ADAM17) is naturally processed through major histocompatibility complex (MHC) class I molecules and is a potential immunotherapeutic target in breast, ovarian and prostate cancers

Abstract

Selection of suitable antigens is critical for the development of cancer vaccines. Most desirable are over-expressed cell surface proteins that may serve as targets for both antibodies and T cells, thus maximizing a concerted immune response. Towards this goal, we characterized the relevance of tumour necrosis factor-α-converting enzyme (ADAM17) for such targeted therapeutics. ADAM17 is one of the several metalloproteinases that play a key role in epidermal growth factor receptor (EGFR) signalling and has recently emerged as a new therapeutic target in several tumour types. In the present study, we analysed the expression profile of ADAM17 in a variety of normal and cancer cells of human origin and found that this protein is over-expressed on the surface of several types of cancer cells compared to the normal counterparts. Furthermore, we analysed the presentation of a human leucocyte antigen (HLA)-A2-restricted epitope from ADAM17 protein to specific T cells established from normal donors as well as ovarian cancer patients. Our analysis revealed that the HLA-A2-restricted epitope is processed efficiently and presented by various cancer cells and not by normal cells. Tumour-specific T cell activation results in the secretion of both interferon-γ and granzyme B that can be blocked by HLA-A2 specific antibodies. Collectively, our data present evidence that ADAM17 can be a potential target antigen to devise novel immunotherapeutic strategies against ovarian, breast and prostate cancer.

Introduction

The science of tumour immunology is based on the premise that antibodies and cytotoxic T lymphocytes (CTL) can distinguish cancer cells from normal cells by recognizing specific antigens expressed differentially by the cancer. Furthermore, these antigens may be incorporated into therapeutic vaccination strategies intended to activate antibody and T cell responses capable of rejecting tumours expressing these antigens. One approach for identifying new antigens for therapeutic vaccines is to exploit differences in the peptides displayed on the cell surface by major histocompatibility complex (MHC) class I molecules [1] in tumours compared to non-malignant cells (reviewed in [2]). Importantly, the proteins from which these peptide epitopes are derived may be those with critical functions in tumour development, growth, survival and metastasis [3]. Indeed, in our search for ovarian cancer antigens, we identified and characterized a number of MHC class I-associated peptides derived from such proteins including a peptide from ADAM metallopeptidase domain 17 (ADAM17)/tumour necrosis factor (TNF)-α-converting enzyme (TACE) [4–6].

ADAM17 is a member of the metalloprotease–disintegrin family of membrane-anchored glycoproteins and is the proteinase responsible for the shedding of the proinflammatory cytokine, TNF-α[7,8] and several epidermal growth factor receptor (EGFR)-binding ligands, including transforming growth factor (TGF)-α, heparin-binding epidermal growth factor (HBEGF) and amphiregulin [9,10]. ADAM17 is up-regulated in a variety of tumours [11–13] and contributes to tumorigenesis, particularly if aberrant activity amplifies shedding of EGFR ligands with known roles in cancer, such as TGF-α, HBEGF and amphiregulin [14–16]. Thus, in recent years, there has been a growing interest in ADAM17 as a new therapeutic target in several EGFR-dependent tumour types. Identification of an MHC processed and presented ADAM17-derived peptide in ovarian and other tumour cells would therefore make it an ideal target for immunotherapy [4–6].

In the present study, we characterized ADAM17 antigen as a potential target for cancer immunotherapy. To facilitate further development of ADAM17-based immunotherapies, we have generated critical data sets to demonstrate ADAM17 over-expression in a variety of tumours and that ADAM17 may serve as an antigen that stimulates anti-tumour immune responses against a variety of tumours.

Materials and methods

Cell lines and primary cells from human tissues

Human ovarian cancer cell lines SKOV3-A2 (kindly provided by Dr Ioannides from MD Anderson Cancer Center) and OVCAR3, human breast cancer cell lines MDA-MB231 and MCF7, human prostate cancer cell line LNCaP and colon cancer cell line Colo205 were obtained originally from the American Type Culture Collection (ATCC, Manassas, VA, USA). SKOV3-A2, OVCAR3, Colo205 and LNCaP were maintained in RPMI-1640 medium (Mediatech, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, GA, USA), l-glutamine (300 mg/ml), non-essential amino acids, penicillin and streptomycin. MDA-MB231 and MCF7 were maintained in Dulbecco's modified Eagle's medium (DMEM) medium supplemented with 10% FBS and other supplements as listed above. All cell lines were maintained at 37°C in a humidified incubator with 5% CO₂. Non-malignant kidney and liver tissues from HLA-A2⁺ human donors were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA, USA). Tissues were digested enzymatically and cell suspensions were generated as per standard methods. Briefly, tissue samples were minced and digested with 2 mg/ml collagenase, 0·1 mg/ml hyaluronidase and 0·15 mg/ml DNAse in DMEM supplemented with 2 × concentration of antibiotics and anti-mycotics (all reagents were obtained from Sigma-Aldrich, St Louis, MO, USA) at 37°C for 3–6 h. Cell suspensions were washed several times with PBS and DMEM supplemented with 10% FBS. Cell viability was assessed by trypan blue exclusion and cells were frozen in 90% FBS and 10% dimethylsulphoxide (DMSO) (Sigma-Aldrich) for future use.

In vitro generation of peptide specific cytotoxic T lymphocytes (CTLs)

Heparinized blood from healthy HLA-A2⁺ donors was purchased from Research Blood Components, LLC (Brighton, MA, USA). Patient blood samples were obtained under International Review Board-approved protocols from women with ovarian cancer undergoing debulking surgery at Duke University Medical Center. Peripheral blood mononuclear cells were purified using lymphocyte separation medium (Mediatech) using differential centrifugation according to standard methods; 20 × 10⁶ cells were plated per well in 2 ml RPMI-1640 medium supplemented with 10% FBS, l-glutamine (300 mg/ml), non-essential amino acids, sodium pyruvate, penicillin and streptomycin (complete medium) in six-well tissue culture plates (BD, Franklin Lakes, NJ, USA) overnight. Non-adherent cells were removed and saved. Plastic adherent cells were pulsed with 50 µg/ml synthetic peptide and 1·5 µg/ml human β2-microglobulin (Sigma-Aldrich) in complete medium for 2 h. Non-adherent cells were added back in 5 ml complete medium supplemented with interleukin (IL)-7 at 5 ng/ml, keyhole limpet haemocyanin (KLH; Sigma-Aldrich) at 5 µg/ml, granulocyte–macrophage colony-stimulating factor (GM-CSF) at 25 ng/ml and IL-4 at 50 ng/ml (all cytokines and growth factors were purchased from Peprotech, Rocky Hill, NJ, USA). Plates were incubated at 37°C in a humidified incubator with 5% CO₂ for 12 days; 2·0 ml medium was removed from each well and fresh complete medium supplemented with 10 U/ml IL-2 for 2 days. T cells were restimulated with CD4/CD8 T cell-depleted autologous monocytes pulsed with synthetic peptide at 10 µg/ml and 1·5 µg/ml human β2-microglobulin in complete medium containing 5 ng/ml IL-7 and 5 µg/ml KLH for 5 days. IL-2 treatment and in vitro restimulation were repeated thrice at the indicated time intervals prior to use of in vitro expanded T cells in enzyme-linked immunospot (ELISPOT) assays.

ELISPOT assays

In vitro-expanded T cells were used as effectors in ELISPOT assays to assess antigen-stimulated interferon (IFN)-γ release or granzyme B release using ELISPOT assay kits (BD-Pharmingen, San Jose, CA, USA), according to the manufacturer's instructions. Typically, a fixed number of various target cells (5 × 10³ cells per well) and effector cells at an effector-to-target ratios of 100–40:1, were used in ELISPOT assays. T2 cells were pulsed with 20 µg/ml synthetic peptides and 1·5 µg/ml human β2-microglobulin in RPMI-1640 medium supplemented with 1% FBS overnight for use as targets in ELISPOT assays. For antibody blocking experiments, target cells were pretreated with purified W6/32 or BB7·2 antibodies (from BD-Pharmingen) at 1:50 dilution for 1 h prior to addition to T cell cultures. ELISPOT assays were performed in replicate wells. Spots were quantitated using an immunospot reader from Cellular Technologies Limited (Shaker Heights, OH, USA). Results are presented as number of IFN-γ-producing cells per 1 × 10⁶ cells. Error bars represent standard error of the mean (s.e.m.) of experimental replicates.

Synthetic peptides

Synthetic peptides corresponding to the HLA-A2-presented ADAM17 epitope (p13-YLIELIDRV) and an influenza A virus epitope derived from the matrix protein (GILGVFTL) were supplied by GenScript Corporation (Piscataway, NJ, USA).

Flow cytometry

Peripheral blood mononuclear cells (PBMCs) obtained from healthy donors were treated with phytohaemagglutinin (PHA) (1 µg/10⁶/ml) for 48 h in culture. THP-1, a monocytic myeloma cell line, was cultured in complete RPMI-1640 media. Activated and non-activated PBMCs, THP-1 cells, normal cells from human liver and kidney and cancer cell lines were treated with a blocking solution [1% normal mouse serum from Sigma and 2% bovine serum albumin (BSA) from Sigma in phosphate-buffered saline (PBS)] on ice for 1 h. Cells were then washed once with PBS and treated with either fluorescein isothiocyanate (FITC)-labelled isotype matched control antibody or ADAM17-specific mouse monoclonal antibody at 1:100 dilution in PBS containing 2% BSA (isotype control antibody was purchased from Invitrogen, Carlsbad, CA, USA and ADAM17 ectodomain-specific monoclonal antibody from R&D Systems, Minneapolis, MN, USA) for 1 h on ice. Cells were washed thrice with PBS and subjected to flow cytometry in Guava flow cytometer (Millipore). Samples were analysed using GuavaSoft software (Millipore).

Immunohistochemistry

Paraffin-coated tissue arrays comprising cancer and matched control tissue sections were purchased from Imgenex (San Diego, CA, USA). All chemical reagents used in this protocol are purchased from Sigma unless noted otherwise. Tissue arrays were stained with anti-ADAM17 antibody and horseradish peroxidase (HRP)-conjugated secondary reagent according to the manufacturer's established procedure and can be found at Imgenex's website (http://www.imgenex.com). Briefly, tissue arrays were first deparaffinized by incubating in a dry oven at 62°C for 1 h. Subsequently slides were dewaxed using xylene and hydrated using ethanol and washed in tap water. Antigen retrieval was achieved by treating slides in citrate buffer (0·01 M, pH 6·0) and microwaving, as suggested by the protocol. Endogenous peroxidase was quenched by treating slides with 3% hydrogen peroxide solution for 6 min. Slides were then blocked with blocking serum (normal mouse serum from Sigma at 1:50) for 30 min and treated with anti-ADAM17 antibody (1:100) for 2 h at room temperature. Slides were washed with PBS and incubated with biotinylated anti-mouse secondary antibody for 30 min at room temperature. Subsequently, slides were washed and incubated with avidin-conjugated HRP (1:100) for 30 min at room temperature. Slides were washed with PBS and developed using 3,3′ diaminobenzidine (DAB) solution for 2 min. Reaction was stopped using tap water. Slides were finally counterstained using Meyer's haematoxylin for 10 s and dehydrated in ethanol. Slides were then cleared using xylene and coverslides were mounted with Permount (Vectastain staining reagents were purchased from Vector Laboratories, Burlingame, CA, USA). Slides were viewed under a fluorescence microscope equipped with a digital camera and micrographs were captured (Nikon Eclipse, TE 800-U).

Real-time quantitative reverse transcription–polymerase chain reaction (qRT–PCR)

Real-time qRT–PCR analysis to determine the expression level of ADAM17 was carried out by SABiosciences (Frederick, MD, USA). RNA isolated from normal liver and kidney tissues (obtained from NDRI) or cancer cell lines (SKOV3-A2, MDA-MB231 and LNCaP) were used for the analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control. Following first-strand synthesis, real-time RT–PCR was carried out using reagents generated by SABiosciences. Data analysis was carried out employing the ΔΔC_t method. Results are presented as fold difference of ADAM17 mRNA expressed in cancer cell lines over the control cells obtained from normal liver tissue.

Results

Presentation of ADAM17-derived epitope p13 by various cancer cell lines

In order to assess the presentation of ADAM17-derived epitope p13 (YLIELIDRV) by cancer cell lines, we generated p13-specific T cells in vitro from healthy HLA-A2⁺ donors. These p13-specific T cells were used as effectors and various cancer cell lines as targets in an overnight ELISPOT assay to quantify IFN-γ release. As shown in Fig. 1, in vitro-generated T cells recognize p13-loaded T2 cells. In addition, p13-specific T cells were tested for recognition and secretion of IFN-γ in response to primary cells obtained from healthy HLA-A2⁺ donor or various cancer cell lines (Fig. 1). Normal liver cells are not recognized by p13-specific T cells appreciably, suggesting that p13 epitope is not generated endogenously in normal cells. However, when pulsed with the synthetic p13 peptide these cells activate T cells readily, demonstrating that these cells are capable of presenting exogenously provided peptide antigen. p13-specific T cells are activated by ovarian cancer cells (SKOV3-A2 and OVCAR3), from which p13 was isolated originally as an HLA-A2 presented peptide [4]. Breast cancer cell lines (MDA-MB-231 and MCF7) also activate p13-specific T cells significantly. Interestingly, prostate cancer cell line LNCaP also activates p13-specific T cells, albeit at a lower level. Other cancer cell lines we tested in culture, including colon cancer (Colo205, Fig. 1) and lung cancer cell lines (data not shown), do not activate p13-specific T cells. Thus, these results demonstrate that ADAM17-derived p13 epitope is presented by ovarian, breast and prostate cancer cells.

Fig. 1.

Generation of T cells specific for the tumour necrosis factor-α converting enzyme (ADAM17)-derived human leucocyte antigen (HLA)-A2-restricted p13 epitope from healthy donors. Peripheral blood mononuclear cells (PBMC) from two healthy HLA-A2⁺ donors were stimulated in vitro with synthetic peptides corresponding to the ADAM17-derived HLA-A2 restricted p13 epitope (YLIELIDRV). These cells were tested in an enzyme-linked immunospot assay using T2 cells loaded with the corresponding synthetic peptide, normal liver cell suspensions obtained from a HLA-A2⁺ healthy donor tissue (with or without exogenously provided p13 peptide) and HLA-A2⁺ cancer cell lines (ovarian cancer cell lines SKOV3-A2 and OVCAR3, breast cancer cell lines MDA-MB-231 and MCF7, a prostate cancer cell line LNCaP and a colon cancer cell line Colo205). Interferon (IFN)-γ-producing cells were quantitated using an immunospot reader. Error bars represent standard error of the mean of experimental replicates.

T cell response towards the ADAM17-derived p13 epitope is HLA-A2-restricted

In order to ascertain the HLA restriction of the presentation of p13 epitope to specific T cells, we extended our IFN-γ ELISPOT analysis to include antibodies specific for pan HLA-A, B, C (W6/32) or HLA-A2 (BB7·2) with cancer targets. Our results indicate that the presentation of p13 is HLA-A2-restricted, as evident from the effective inhibition of IFN-γ release by BB7·2 antibody (Fig. 2, top panel). As expected, W6/32 also blocked T cell activation. T2 cells or primary liver cells from an HLA-A2⁺ donor pulsed with an irrelevant peptide (influenza A matrix protein-derived peptide) were used as negative controls, and these cells did not activate p13-specific T cells above background levels. However, as expected, when pulsed with p13 peptide, these cells activated p13-specific T cells readily. These results demonstrate that the p13 peptide-specific T cells are highly specific and the presentation of p13 peptide to these T cells is HLA-A2-restricted.

Fig. 2.

T cells specific for the tumour necrosis factor (TNF)-α converting enzyme (ADAM17)-derived p13 epitope are both human leucocyte antigen (HLA)-A2-restricted and cytolytic. Peripheral blood mononuclear cells (PBMC) from a healthy HLA-A2⁺ donor were stimulated in vitro with synthetic peptides corresponding to the ADAM17-derived p13 epitope (YLIELIDRV). (a) These cells were tested in an enzyme-linked immunospot (ELISPOT) assay using T2 cells or normal kidney cells obtained from an HLA-A2⁺ healthy donor tissue loaded with an irrelevant synthetic peptide (flu) or p13 peptide. HLA-A2⁺ cancer cell lines (ovarian cancer cell line SKOV3-A2 and breast cancer cell line MDA-MB-231) were pretreated with W6/32 or BB7·2 antibody prior to incubation with p13 specific T cells overnight. Interferon (IFN)-γ-producing cells were quantitated using an immunospot reader. (b) p13-specific T cells were used in an ELISPOT assay with the above-mentioned targets and the release of granzyme B was measured as described above. Error bars represent standard error of the mean of experimental replicates.

Cytolytic potential of the T cells specific for the ADAM17-derived p13 epitope

Although we observed that the p13-activated T cells could recognize p13 epitope-expressing tumour cells, we wished to determine if these p13-activated T cells were cytolytic by measuring the release of granzyme B in an ELISPOT assay in response to various targets. Results demonstrate that a significant portion of p13-activated T cells indeed secrete granzyme B in response to p13 presentation by p13 peptide-loaded T2, normal liver cells or various cancer targets, but not T2 cells or normal liver cells pulsed with a negative control peptide (Fig. 2, bottom panel). This secretion could be blocked significantly by the inclusion of W6/32 or BB7·2 antibodies, thus demonstrating the HLA-A2-restricted peptide presentation.

Generation of ADAM17-derived epitope p13-specific T cells from ovarian cancer patients

Our next question was to determine if T cells can be activated from PBMC obtained from HLA-A2⁺ ovarian cancer patients using p13 synthetic peptide. IFN-γ ELISPOT assays using ovarian cancer patient T cells stimulated in vitro with p13 peptide and various targets demonstrate that p13 peptide-specific T cells can be activated from ovarian cancer patients, and they recognize peptide-pulsed T2 but not normal liver cells (Fig. 3). These T cells also recognize ovarian (SKOV3-A2 and OVCAR3) and breast (MDA-MB231) cancer cell lines. These results demonstrate that functional p13 peptide-specific T cells can be generated from ovarian cancer patients.

Fig. 3.

Generation of T cells specific for the tumour necrosis factor-α-converting enzyme (ADAM17)-derived human leucocyte antigen (HLA)-A2-restricted p13 epitope from ovarian cancer patients. Peripheral blood mononuclear cells (PBMC) from two HLA-A2⁺ patients with ovarian cancer were stimulated in vitro with the synthetic peptide corresponding to p13 peptide (YLIELIDRV). These cells were tested in an enzyme-linked immunospot assay using T2 cells loaded with the corresponding synthetic peptide, normal liver cell suspensions obtained from a HLA-A2⁺ healthy donor tissue (with or without exogenously provided p13 peptide) and HLA-A2⁺ cancer cells lines (breast cancer cell line MDA-MB-231 and ovarian cancer cell lines OVCAR3 and SKOV3-A2). Interferon (IFN)-γ producing cells were quantitated using an immunospot reader. Error bars represent standard error of the mean of experimental replicates.

ADAM17 is over-expressed on the surface of a variety of cancer cells

In order to evaluate the expression levels of ADAM17 on the surface of normal and cancer cells, we performed flow cytometry employing a ADAM17-specific antibody that recognizes the ectodomain of ADAM17 protein. As shown in Fig. 4a, primary cells prepared from healthy human liver tissue do not express detectable levels of ADAM17. Primary cells from a healthy human kidney tissue express low levels of ADAM17 on their surface. However, ADAM17 expression is detectable at higher levels in ovarian (SKOV3-A2 and OVCAR3), breast (MDA-MB-231 and MCF7), prostate (LNCaP) and colon (Colo205) cancer cell lines. Notably, SKOV3-A2 and LNCaP express very high levels of ADAM17. These results demonstrate that ADAM17 is expressed on the surface of a variety of cancer cells, but its expression on normal cells is undetectable to extremely low. ADAM17 is involved in the release of TNF-α from activated PBMCs and monocytes. However, expression levels of TACE in these cell types have never been reported. Because we are postulating that TACE could be a potential immunotherapeutic antigen, the question of expression levels in these critical cell types needed to be addressed. Hence, we performed flow cytometry analysis of activated PBMCs and THP-1 (myelomonocytic cell line) to determine the levels of TACE in these cell types. As shown in Fig. 4b, the expression levels of ADAM17 were low in PHA-activated and non-activated PBMCs as well as THP-1 cells compared to ovarian tumour cell line SKOV3 and other tumour cell lines (Fig. 4a), demonstrating that ADAM17 can be a potential immunotherapeutic target.

Fig. 4.

Analysis of surface expression of tumour necrosis factor-α converting enzyme (ADAM17) in normal and cancer cells by flow cytometry. (a) Normal cell suspensions (liver and kidney) obtained from healthy human leucocyte antigen (HLA)-A2⁺ donors or cancer cell lines (ovarian cancer cell lines SKOV3-A2 and OVCAR3, breast cancer cell lines MDA-MB-231 and MCF7, prostate cancer cell line LNCaP and a colon cancer cell line, Colo205) were treated with either fluorescein isothiocyanate (FITC-labelled isotype-matched control antibody or FITC-labelled ADAM17-specific antibody and subjected to flow cytometry by Guava flow cytometer (Millipore). Samples were analysed using GuavaSoft software (Millipore). (b) SKOV3-A2, peripheral blood mononuclear cells (PBMC), PBMC treated with phytohaemagglutinin (PHA) and human acute monocytic leukaemia cell line (THP-1) cells were stained with either FITC-labelled isotype matched control antibody or FITC-labelled ADAM17-specific antibody and subjected to flow cytometry as described above.

ADAM17 over-expression in cancer tissues but low expression in normal tissues

We then assessed ADAM17 expression levels in cancer tissues. We chose to perform immunohistochemical analysis on tissue sections from ovarian, breast and prostate cancers and tissue-matched non-malignant controls. Results of our analysis are shown in Fig. 5. From the micrographs, it is evident that ADAM17 is expressed at very high levels in cancer tissues, but its expression is low in normal tissues. These results show that ADAM17 is not only over-expressed in cancer cell lines, but also in primary cancer tissues isolated from patients.

Fig. 5.

Immunohistochemical analysis of expression of tumour necrosis factor (TNF)-α converting enzyme (ADAM17) in normal and cancer cells. Tissue arrays comprising of matched normal or cancer specimens (breast, ovarian and prostate tissues) were treated with an ADAM17-specific monoclonal antibody followed by treatment with a horseradish peroxidise (HRP)-conjugated secondary antibody reagent (detailed protocol is described in Materials and methods section). Slides were developed with 3,3′ diaminobenzidine (DAB) substrate and tissues were counterstained with Meyer's haematoxylin. Stained tissue sections were analysed using a fluorescent microscope and micrographs were captured at 200× magnification. (a and b) Breast normal and cancer, (c and d) ovarian normal and cancer, (e and f) prostate normal and cancer.

ADAM17 mRNA expression does not correlate with increased ADAM17 protein expression

In order to determine if increased expression of ADAM17 is regulated at the transcriptional level, we performed real-time qRT–PCR analysis, a powerful tool to determine the transcript levels of a given gene. We used RNA isolated from normal liver and kidney cells as controls. ADAM17 mRNA level is elevated slightly in SKOV3-A2 compared to normal liver and kidney cells (Fig. 6). However, the levels are reduced in MDA-MB-231 and LNCaP cells. These data support the notion that elevated ADAM17 expression in cancer cells is regulated at the translational or post-translational, but not the transcriptional level.

Fig. 6.

Expression level of tumour necrosis factor-α-converting enzyme (ADAM17) in normal and cancer cells by real-time quantitative reverse transcription–polymerase chain reaction (qRT–PCR) analysis. Real-time qRT–PCR analysis to determine the expression levels of the genes that code ADAM17 was carried out using RNA isolated from normal tissues (liver and kidney) or cancer cell lines (ovarian cancer cell line SKOV3-A2, breast cancer cell line MDA-MB-231 and prostate cancer cell line LNCaP). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control. Data analysis was carried out employing the ΔΔC_t method. Results are presented as fold difference of ADAM17 expression in cancer cell lines over the control cells obtained from normal liver tissue.

Discussion

It is well established that human cancers express antigens that can be targeted specifically by cell-mediated and humoral immunity [17–20]. However, which antigens contain epitopes that can serve as authentic T cell rejection targets is largely unknown. We used the MHC class I-associated peptide repertoire in tumour cells as a source for identifying new tumour antigens for the development of cancer vaccines. Although a number of tumour antigens have been identified for potential use in cancer vaccines, it is essential to characterize them using various biological criteria for their translational applicability. In this study, we have characterized in depth the tumour-associated antigen ADAM17, which was discovered by analysing the MHC class I peptide repertoire in ovarian cancer cells [4]. ADAM17 protein is up-regulated and contributes to tumorigenesis in a variety of tumours [11,12,15] and has been well studied as a drug target [11,21]. However, ADAM17 as a cancer immunotherapy antigen has not been validated previously.

Herein, we report the characteristics of ADAM17 that make it suitable for both cell-mediated and humoral clinical cancer immunotherapy applications. Specifically, we have demonstrated that an HLA-A2-restricted peptide epitope of ADAM17 is capable of inducing T cell responses which can recognize ovarian, breast and prostate cancers in an HLA-A2 restricted fashion. The peptide-specific T cells not only secrete IFN-γ in response to the antigen stimulation but also secrete granzyme B, demonstrating the cytolytic potential of these in vitro-generated T cells. The fact that the ADAM17-epitope specific CTL could be generated from cancer patients in addition to healthy donors suggests that precursor T cells specific for ADAM17 are normally present. Flow cytometry and immunohistochemistry demonstrating that this protein is expressed abundantly on the surface of ovarian, prostate and breast cancer cell lines and patient tissues, but not on their normal counterparts, also signifies the possible use of this antigen as an antibody target. Future studies will use the serum of mice vaccinated against ADAM17 to assess for the ability of vaccine-induced antibodies to bind to surface-expressed ADAM17 and to determine the functional consequences of this binding.

One possible criticism of immunotherapeutic targeting of ADAM17, also called TNF-α-converting enzyme (TACE), is that it is involved in cleavage of TNF-α from activated T cells and monocytes [7] and thus T cells expressing the molecule might be susceptible to immune attack. It was therefore critical to assess the expression levels of ADAM17 on activated PBMC. Our flow cytometry data, using the antibody that binds to the ectodomain of ADAM17, indicates low level of expression in PHA-activated and non-activated PBMCs and a monocytic cell line, THP-1, comparable to normal tissues that we have tested (Fig. 4b). The localization of ADAM17 is speculated to be an important determinant of shedding activity. TNF-α processing has been shown to occur in the trans-Golgi network, and be connected closely to transport of soluble TNF-α to the cell surface. However, several studies suggest that the majority of mature, endogenous ADAM17 may be localized to a perinuclear compartment, with only a small amount being present on the cell surface. This perinuclear localization of ADAM17 indicates the possibility that ADAM17-mediated TNF-α shedding may also occur in the intracellular environment [22,23] suggesting less likelihood of an adverse effect on lymphoid cells caused by immunotherapeutic targeting of surface over-expression in tumours.

Our approach of antigen identification by MHC class I peptide repertoire analysis differs from the more common differential gene expression analysis of normal and cancer tissues [24,25]. However, gene expression may not reflect the protein level or stability and modifications of the protein that are relevant to immunotherapy [26–28]. As indicated by Chiriva-Internati et al., it is critical to validate candidate antigens for clinical applications using a system level-based approach at the level of cells, tissues and organs beyond gene expression [29]. Indeed, our real-time qRT–PCR analysis (Fig. 6) revealed that the transcript levels of ADAM17 in cancer cells do not reflect the enhanced protein expression as detected by flow cytometry and immunohistochemistry. Furthermore, one important observation was that tumours that had high expression of ADAM 17 protein were not always the most sensitive to T cell-mediated killing, as demonstrated by the LNCaP and Colo205 data (Figs 1 and 4a). It should be noted that although protein over-expression is a prerequisite for antibody or drug-mediated targeting, it is not a critical factor for MHC class I processing and presentation of the T cell epitope, which correlates primarily with the level of degradation of proteins associated with misfolding, cryptic translation and other causes of high turnover [1,30] which occur frequently in cancer initiation, survival and progression [2].

In summary, the characterization of ADAM17 as an immunotherapy target suggests that vaccine strategies utilizing it should proceed. Indeed, the T cell epitope derived from ADAM17 has been evaluated for safety and CTL responses in patients with breast and ovarian cancers in a Phase I clinical study (Morse, Alvarez Secord and Philip, manuscript in preparation). Furthermore, because ADAM 17 is the principal sheddase for TGF-α and EGFR-ligands from a late secretory pathway compartment [31], it may be possible to combine small molecules inhibiting the EGFR pathway in conjunction with immunotherapy against ADAM17 as a combination therapy.

Disclosure

The authors have no disclosures to declare.