Six-transmembrane epithelial antigen of the prostate and enhancer of zeste homolog 2 as immunotherapeutic targets for lung cancer

Background T-cell based immunotherapy for lung cancer (LC) could be a promising and novel therapeutic approach. Six-transmembrane epithelial antigen of the prostate (STEAP) and the polycomb group protein enhancer of zeste homolog 2 (EZH2) are highly expressed in LC and since the expression of molecules in normal tissue is significantly lower as compared to tumor cells, these proteins are considered as potential tumor-associated antigens (TAAs) for developing T-cell based immunotherapy. Methods We assessed the capacity of predicted CD4 T-cell epitopes from STEAP and EZH2 to induce anti-tumor immune responses to LC cell lines. Results Out of several predicted epitopes, two synthetic peptides, STEAP281-296 and EZH295-109, were effective in inducing CD4 T-cell responses that were restricted by HLA-DR1, DR15, or DR53 molecules, indicating that the peptides function as promiscuous T-cell epitopes. Moreover, STEAP281-296 and EZH295-109-reactive T-cells could directly recognize STEAP or EZH2 expressing LC cells in an HLA-DR restricted manner. In addition, some STEAP-reactive T-cells responded to STEAP+ tumor cell lysates presented by autologous dendric cells. Most significantly, both of these peptides were capable of stimulating in vitro T-cell responses in patients with LC. Conclusions Peptides STEAP281-296 and EZH295-109 function as strong CD4 T-cell epitopes that can elicit effective anti-tumor T-cell responses against STEAP or EZH2 expressing LC. These observations may facilitate the translation of T-cell based immunotherapy into the clinic for the treatment of LC.


Background
Lung cancer (LC) represents a significant health problem with 222,520 new cases and 157,300 deaths in the past 10 years in the United States [1]. Recently, adjuvant cisplatin-vinorelbine chemotherapy in completely resected non-small cell LC (NSCLC) has resulted in an enhanced survival benefit at 5 years (8.9% improvement versus observation) [2]. Molecular target-based drugs (gefitinib, erlotinib, etc.) are available for specific types of NSCLC showing epidermal growth factor receptor mutations. However, these chemotherapeutic regimens can be extremely toxic and provide limited survival benefit for advanced LC. Thus, the development of novel and less toxic alternatives such as immunotherapy is warranted. Nevertheless, the success of immunotherapy will ultimately rely on the identification of appropriate tumor-associated antigens (TAAs) that are overexpressed in tumor cells relative to normal tissues.
Six-transmembrane epithelial antigen of the prostate (STEAP) is a 339 amino acid protein that is critical for erythroid iron homeostasis. STEAP is located on the cell surface [3] and is predominantly overexpressed in various tumor types (prostate, bladder, colon, ovarian, and Ewing sarcoma) [4]. The enhancer of zeste homolog 2 (EZH2) is a polycomb group protein that functions as a regulator of homeobox gene expression [5]. EZH2 is highly expressed in various tumor types including prostate [6], breast [7], esophagus [8], and pancreatic [9] cancers. Moreover, the expression of EZH2 has been linked to tumor aggressiveness and metastatic potential, and has been linked to a poor overall patient prognosis [6][7][8]. The low expression of STEAP and EZH2 in normal tissues together with recent studies reporting that these molecules are overexpressed in NSCLC, suggests that both protein could be utilized as TAAs in LC [10,11].
Although CD8 cytotoxic T lymphocytes are believed to have a major role in eradicating cancer, CD4 helper T lymphocytes are likely to have a critical role in immunotherapy since they participate in generation and persistence of CD8 T-cell responses [12]. In addition, CD4 T-cells exhibit an effector role against tumors that express HLA-DR molecules [13]. For the development of peptide-based immunotherapies against LC, we have searched for possible HLA-DR epitopes capable of eliciting CD4 T-cell responses to STEAP and EZH2. Here, we report that 2 epitopes, STEAP 281-296 and EZH2 95-109 were capable of eliciting in vitro antigen-specific, HLA-DR-restricted CD4 T-cell responses against LC cells expressing STEAP and EZH2. In addition, peptides STEAP 281-296 and EZH2 95-109 were also found to stimulate T-cell responses in LC patients. We believe that these results may be of use for the development of Tcell based immunotherapy for LC.

Western blotting
One million cells were washed in phosphate buffered saline (PBS) and lysed in NuPAGE LDS sample buffer (Invitrogen). The cell lysate was subjected to electrophoresis in a 4-12% NuPAGE bis-Tris SDS-PAGE gel (Invitrogen) under reducing condition and transferred to Immobilon-P (Millipore, Bedford, MA, USA) membrane. The membrane was blocked in PBS containing 0.01% Tween 20 and 5% non-fat dry milk for 1 h at room temperature and incubated with rabbit anti-human STEAP polyclonal antibody (H-105, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:200 in blocking buffer, anti-human EZH2 mouse monoclonal antibody (mAb) (BD.612666, BD Bioscience, San Jose, CA, USA) diluted 1:200, or antiβ-actin mAb (C4, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:1,000 in blocking buffer as the control, for 18 h at 4°C. After washing, the membrane was incubated with horseradish peroxidase-labeled sheep anti-rabbit or -mouse IgG and subjected to the enhanced chemiluminescence assay using the ECL detection system (Amersham, Buckinghamshire, UK).

In vitro induction of antigen-specific CD4 T-cell clones with synthetic peptides
The procedure utilized for the generation of STEAP and EZH2 reactive CD4 T-cell clones using peptide-stimulated lymphocytes from peripheral blood mononuclear cells (PBMCs) of human healthy individuals has been described in detail [16]. Briefly, dendritic cells (DCs) were produced from purified CD14 monocytes (using antibody-coated magnetic microbeads from Miltenyi Biotech, Auburn CA, USA) cultured for 7 days at 37°C in a humidified CO 2 (5%) incubator in the presence of 50 ng/ml GM-CSF and 1,000 IU/ml IL-4. Peptide-pulsed DCs (3 μg/ml for 2 h at room temperature) were irradiated (4,200 rad) and co-cultured with autologous purified CD4 T-cells in 96-flat-bottomed-well culture plates. One week after peptide stimulation, the CD4 T-cells were restimulated in individual microcultures with peptide-pulsed irradiated autologous PBMCs and 2 days later, recombinant human IL-2 was added at a final concentration of 10 IU/ml. One week later, the T-cells were tested for antigen reactivity using a cytokinerelease assay as described below. Those microcultures exhibiting a significant response of cytokine-release to peptide (at least 2.5-fold over background) were cloned by limiting dilution and expanded in 24-well plates by weekly restimulation with peptides and irradiated autologous PBMCs. Complete culture medium for all procedures consisted of AIM-V medium (Invitrogen/GIBCO, Carlsbad, CA, USA) supplemented with 3% human male AB serum. All blood samples were obtained after the appropriate informed consent.

Measurement of antigen-specific responses with established CD4 T-cell clones
CD4 T-cells (3 × 10 4 /well) were mixed with irradiated antigen-presenting cells (APCs) in the presence of various concentrations of antigen (peptides, tumor lysates) in 96-well culture plates. APCs consisted of autologous PBMCs (1 × 10 5 /well), HLA-DR expressing L-cells (3 × 10 4 well), autologous DCs (5 × 10 3 /well) or LC cell lines (3 × 10 4 /well). The LC cell lines were previously treated with IFN-γ at 500 U/ml for 48 h to enhance HLA-DR expression. The expression of HLA-DR molecules on tumor cells was evaluated by flow cytometry using anti-HLA-DR mAb conjugated with fluorescein isothiocyanate (BD Pharmingen, San Diego, CA). Tumor cell lysates were prepared by 3 freeze-thaw cycles of 1 × 10 8 tumor cells, resuspened in 1 ml of serum-free AIM-V medium. Tumor cell lysates served as an antigen at 5 × 10 5 cell equivalents/ml. Culture supernatants were collected after 48 h for measuring antigen-induced lymphokine (IFN-γ or GM-CSF) or granzyme B production by the CD4 T-cells using ELISA kits (IFN-γ and GM-CSF; BD Pharmingen, San Diego, CA, USA, granzyme B; Mabtech AB, Nacka Strand, Sweden). To demonstrate antigen-specificity and HLA-DR restriction, blocking of antigen-induced responses were assessed by adding anti-HLA-DR mAb L243 (IgG2a prepared from supernatants of the hybridoma HB-55 obtained from the ATCC) or the anti-HLA-A/B/C mAb W6/32 (IgG2a, ATCC) at 10 μg/ml throughout the 48 h incubation period.

Cell-mediated cytotoxicity assays
Cytotoxic activity of CD4 T-cell clones was measured using a colorimetric CytoTox 96 assay (Promega, Madison, WI, USA). This system quantifies the release of lactate dehydrogenase (LDH) from target cells. T-cells were mixed with 2 × 10 4 target cells at different effector to target (E:T) ratios in 96-round-bottomed-well plates. After 6 h incubation at 37°C, 50 μl supernatant samples were collected from each well to measure LDH content.

Measurement of peptide-specific responses in LC patients
PBMCs were isolated from fresh heparinized blood by Ficoll-Hypaque centrifugation and washed twice with RPMI 1640. PBMCs were resuspended in complete medium and placed at 1.5 × 10 5 cells/well and cultured in triplicate in 96-round-bottomed well plates in the presence of 10 μg/ml peptide. Negative controls (in the absence of peptide), were done in eight replicate samples. One week later, the cultures were restimulated with peptide-pulsed (10 μg/ml) irradiated autologous PBMCs (5 × 10 4 /well). After 7 days of restimulation, supernatants were harvested for examining the ability of peptide-induced lymphokine production by the LC patient's PBMCs. The institutional ethics committee had approved the study protocol and the appropriate informed consent for blood donation was obtained from all patients before blood sampling.

Expression of STEAP and EZH2 in LC samples and tumor cell lines
In immunohistochemistry studies with several surgical specimens of LC, we observed moderate to strong staining with anti-STEAP antibody and anti-EZH2 mAbs. Conversely, staining of adjacent normal tissues with these antibodies was much weaker. STEAP was detected in 8/8 cases of the adenocarcinoma and 6/6 squamous cell carcinoma cases. EZH2 was detected in 5/5 cases of the adenocarcinoma and in 5/5 squamous cell carcinoma cases (examples are shown in Figure 1). In addition, we performed immunohistochemical staining for expression of HLA-DR in 5 lung adenocarcinomas and 4 lung squamous cell carcinomas. We have found the expression of HLA-DR in the adenocarcinoma cells (4/5 cases) and in the infiltrating APCs, lymphocytes and alveolar macrophages ( Figure 2). These observations suggest that tumor cells could be logical targets of STEAP-or EZH2-specific CD4 T-cells in situ.
Western blot analysis revealed that STEAP was present in several tumor cell lines including LCs (except  for Jurkat T cell lymphoma, which served as a negative control), but was absent in PBMC samples from healthy individuals ( Figure 3A). EZH2 was detected in all the tumor cell lines (including Jurkat), but not in PBMCs ( Figure 3B). These observations provide a rationale for exploring the use of STEAP and EZH2 as TAAs for developing anti-tumor T-cell based immunotherapy for LC (and the other tumor types).
Selection of potential HLA-class II restricted peptide epitopes from EZH2 and STEAP To identify the promiscuous (broadly degenerate) HLAclass II binding peptide epitopes is very important since using the degenerate epitope-based vaccines in applicable to general population. For predicting promiscuous HLA-class II binding peptides, we used computer-based MHC peptide binding motifs for three common HLAclass II molecules, HLA-DR1, DR4 and DR7 [14]. In addition, this algorithm indicate that some peptides that score high for the DR1, DR4 and DR7 alleles also have the capacity to bind to additional HLA-class II alleles such as DR9, DR13, DR15 and DR53. Therefore, we have reported that some peptides predicted by additional HLA-class II alleles, such as DR9, DR13, DR15 or DR53 [17]. In this study, the prediction system could select nine peptide sequences from EZH2 (data not shown) and we selected the three highest potentially promiscuous peptides, EZH2 95-109 , EZH2 220-234 and EZH2 693-704 . In the case of STEAP, the algorithm system suggested 28 peptide sequences (data not shown) as potentially promiscuous HLA-class II binders and we have reported that two STEAP peptides, STEAP 102-116 and STEAP 192-206 were effective in stimulating in vitro anti-tumor T helper responses [18,19]. In the present study, considering that an ideal TAA should contain peptide epitopes to stimulate both CD8 and CD4 T-cells, we selected 2 STEAP peptides from these promiscuous binders, STEAP 261-275 , which overlaps with HLA-A2-restricted CD8 T-cell epitope STEAP 262-270 [20] and STEAP 281-296 , which lies proximal to HLA-A2-restricted CD8 epitope, STEAP 292-300 [21]. We synthesized the three EZH2 and two STEAP epitope peptides and proceeded to determine whether these peptides could induce CD4 helper T-cell responses in vitro.

Induction of CD4 T-cell responses to STEAP and EZH2 peptide epitopes
Next, we evaluated whether these peptides were capable of stimulating CD4 T-cells obtained from 6 healthy individuals (HLA-DR4/15, HLA-DR4/9, HLA-DR4/15, HLA-DR1/15, HLA-DR9/13 and HLA-DR9/14) using autologous DCs as APCs. After 2-3 peptide restimulation cycles, 2 of 5 peptides (STEAP 281-296 and EZH2 95-109 ) were able to induce peptide-specific CD4 T-cell responses. To carry out more detailed antigen specificity and HLA-DR restriction analyses, and to assess tumor cell recognition, T-cell clones (4 reactive with STEAP 281-296 and 6 reactive with EZH2 95-109 ) were isolated by limiting dilution. As shown in Figure 4, all of these clones recognized their respective peptide epitopes in a dose dependent manner and this recognition was inhibited by anti-HLA-DR (L243) mAb but not by anti-HLA-class I (W6/32) mAb. Since mAb L243 is specific for HLA-DR, these results indicate that the presentation of STEAP 281-296 and EZH2 95-109 to the T-cells is via HLA-DR molecules and not via other HLA-class II molecules (HLA-DQ or DP). To more specifically delineate the HLA-DR restriction alleles, we studied the T-cells responses using a panel of HLA-DR transfected mouse L-cells and HLA-typed human EBV-LCLs. As shown in Figure 5A, the STEAP 281-296 -reactive clone SH31 was restricted by HLA-DR15 molecules, and clones SH6, HK7 and Sa12 recognized the same peptide but in the context of HLA-DR53. The EZH2 95-109 epitope was presented to clone SK31 via HLA-DR1, to clones SK32 and Sa361 by HLA-DR15 and to clones MT10, Sa362 and YM18 by HLA-DR53 ( Figure 5B, C and 5D). These results indicate that STEAP 281-296 and EZH2 95-109 can be presented to CD4 T-cells by more than one HLA-DR allele, behaving as classical promiscuous epitopes.

Recognition of naturally processed exogenous antigen by STEAP-reactive CD4 T-cell clones
In addition, we assessed whether APCs such as DCs could capture and process antigens derived from dead STEAP expressing tumor cells (freeze-thaw cell lysates) and present the STEAP epitope to the CD4 Tcell clones. As shown in Figure 9, the STEAP 281-296reactive CD4 T-cell clones SH31, SH6 and Sa12 recognized naturally processed epitope presented by DCs derived from STEAP positive LC cell lysates (LU65, A549, RERF-LC-AI) but not STEAP negative, Jurkat cell lysates. These responses were inhibited by L243 mAb (anti-HLA-DR) but not by W6/32 mAb (anti-HLA-class I), indicating that CD4 T-cell responses were antigen-specific and via the interaction of T-cell receptor with HLA-DR molecules. These results indicate that the STEAP 281-296 epitope can be presented to T-cells via either the endogenous (direct tumor recognition) or exogenous (DCs) antigen processing pathways. Conversely, the EZH2-reactive CD4 T-cell clones were unable to recognize naturally processed epitopes derived from tumor cell lysates via DCs (data not shown).

STEAP and EZH2 peptide recognition by PBMCs from patients with LC
Lastly, to further evaluate the capacity of LC patients to exhibit CD4 T-cell responses to STEAP 281-296 and EZH2 95-109 , we stimulated PBMCs from 10 LC patients and 3 healthy donors with the corresponding peptides in short-term cultures. Seven days after the second stimulation, culture supernatants were collected and IFNγ or GM-CSF production was assessed. Peptide TT 830-843 was used as a positive control since this antigen has ability to induce strong CD4 T-cell responses in the majority individuals regardless of their HLA-DR alleles. The results presented in Table 1 show significant T-cell responses to STEAP 281-296 in 9/10 patients, to EZH2 95-109 in all patients, and to the positive control TT 830-843 in all patients. These results suggest that T-cell precursors that are reactive with the STEAP and Discussion T-cell based immunotherapy has become an accepted treatment alternative for cancer. The presence of lymphocytic infiltrations in murine and human lung tumors suggests that an immune reaction could potentially help to control LC progression [22,23]. In view of this, we believe that identifying TAAs and the corresponding peptide epitopes capable of eliciting anti-tumor T-cell responses will be critical to develop peptide-based cancer immunotherapy. EZH2 plays a role in cell cycle regulation and proliferation and increased EZH2 expression has been correlated with aggressive tumor behavior and poor survival in LC [18]. STEAP is overexpressed in various types of tumors including LC. In addition, it has been reported that generation of CD4 or CD8 T lymphocytes recognizing STEAP and EZH2 indicates that STEAP and EZH2 could be potent TAAs for T-cell based immunotherapy [18][19][20][21]24].
The goal of the present study was to assess whether STEAP and EZH2 could function as TAAs for LC, capable of generating anti-tumor CD4 T-cell responses. The results showed that two peptides, STEAP 281-296 and EZH2 95-109 , were efficient in activating and expanding CD4 T-cells obtained from healthy individuals and LC patients. The results show that STEAP 281-296 could be presented to CD4 T-cells by either HLA-DR15 or DR53 and that EZH2 95-109 is presented in the context of DR1, DR15 or DR53 ( Figure 5). The HLA-DR53 molecule is expressed by a large proportion of the population (~50%) owing to its linked co-expression with the HLA-DR4, DR7, and HLA-DR9 alleles [14]. Thus, each peptide would provide broad coverage for clinical use. Most significant was the demonstration that the CD4 T-cell clones generated by STEAP 281-296 and EZH2 95-109 were capable of recognizing naturally processed antigen directly on HLA-DR expressing tumor cells and that the T-cells exhibited cytotoxicity against the tumor cells. These findings suggest the possibility of direct therapeutic benefit of CD4 anti-tumor T-cell responses independent of CD8 T-cells. Both STEAP 281-296 and EZH2 95-109  epitope peptides were able to stimulate T-cell responses in LC patients indicating that T-cell precursors capable of recognizing STEAP 281-296 or EZH2 95-109 exist in both healthy individuals and some patients with LC.
As with the many TAA such as CEA, PSA or Her2/ neu, message and protein levels of EZH2 or STEAP appear to be low amounts in normal tissues. Our results show that normal cells such as PBMCs and DCs were not recognized by the STEAP or EZH2-reactive CD4 Tcells. Furthermore, Western blot analysis revealed no detectable STEAP or EZH2 expression in PBMCs and no significant expression of STEAP or EZH2 was found in normal tissues using immunohistochemical staining. Thus, we believe that no significant autoimmune pathology would be elicited if STEAP or EZH2 CD4 T-cell epitopes were used for cancer immunotherapy. Nevertheless, it would be wise to assess in more detail the levels of STEAP or EZH2 in other normal tissues (e.g., liver, kidney, brain) before setting about clinical trials using STEAP or EZH2 as targets for immunotherapy.
It is possible that direct stimulation of T-cells, even priming by tumor cells may be actually tolerant toward TAAs. In fact, EZH2 and STEAP express in very low amounts in normal tissues and benign neoplasms and upregulated and overexpressed in malignant tumors. These low-and high-level expressions may induce tolerance by deletion of the TAA-specific T-cell repertoire, especially T-cells with high affinity for its peptide epitope. However, it appears that tolerance induction by the tumors expressing, STEAP or EZH2 may not be leaded, because we observed that cancer patient's PBMC could respond to the peptides and produced lymphokines. These results suggest that the STEAP-or EZH2reactive T-cells are not tolerant and completely deleted by tumor cells in vivo.
The present study is the first report of the identification of an immunogenic epitope of EZH2 recognized by CD4 T-cell and suggests that EZH2, which is the overexpressed TAA in LC, could be significant immunogenic target for inducing both CD4 and CD8 T-cell against LC. In addition, we present new CD4 T-cell epitope STEAP 281-296 , which is closely located in the HLA-A2 restricted CD8 T-cell epitope, STEAP 292-300 [21]. Wroblewski et al demonstrated that some LC cell lines (23%) constitutively express HLA-DR molecules and treatment of IFN-γ could enhance expression of HLA-DR molecules in LC cells [25]. Another published studies described that 45% of lung adenocarcinoma  specimens expressed HLA-DR [26] and HLA-DR molecules on primary tumors were detected in 10% of patients with micrometastasis to regional lymph nodes and in 30% of patients without tumor spread [27]. We were also able to verify that the expression of HLA-DR was found in some lung adenocarcinomas and tumor adjacent infiltrating APCs (Figure 2). These observations suggest that CD4 T-cells have important role in immunological response to LC and tumor cells are logical targets of the TAA-specific CD4 T-cells in vivo. In addition, the predicted peptide STEAP 281-296 and EZH2 95-109 could be capable of stimulating T-cell responses in some LC patients. Taken together these results, we believe that the simultaneous induction of STEAP-and EZH2-specific CD4 and CD8 T-cell responses would be more effective in achieving antitumor effects than the separate induction of CD4, or CD8 T-cell responses. Moreover, the recent development of EZH2 inhibitors as anti-cancer therapeutics [9] opens the interesting opportunity of a combined chemotherapy/immunological approach for treating EZH2expressing LCs.

Conclusions
Peptides STEAP 281-296 and EZH2 95-109 are newly described CD4 T-cell epitopes that can elicit effective anti-tumor T-cell responses against STEAP or EZH2 expressing LC cell lines. These peptides may facilitate the development of peptide-based vaccines for LC.