CN105163754A - Prostate-specific tumor antigen and uses thereof - Google Patents

Abstract

Twenty-one PSGR-derived peptides predicted by an immuno-informatics approach based on the HLA-A2 binding motif were examined for their ability to induce peptide-specific T cell responses in peripheral blood mononuclear cells (PBMCs) obtained from either HLA-A2+ healthy donors or HLA-A2+ prostate cancer patients. The recognition of HLA-A2 positive and PSGR expressing LNCaP cells was also tested. Three peptides, PSGR3, PSGR4 and PSGR14 frequently induced peptide-specific T cell responses in PBMCs from both healthy donors and prostate cancer patients, and are recognized by CD8+ T cells in an HLA-A2 dependent manner. These peptide-specific T cells recognize HLA-A2+ and PSGR+ tumor cells, and killed LNCaP prostate cancer cells in an HLA class I-restricted manner. These PSGR-derived peptides identified are useful as diagnostic markers as well as immune targets for anticancer vaccines.

Description

Prostate-specific tumor antigen and uses thereof

Background technique

Prostate cancer has become the most common cancer and the second leading cause of cancer death in American men [1]. The standard of care for most men with prostate cancer is surgery and/or radiation therapy. However, disease recurrence occurs in up to 30% of patients after surgery or radiotherapy. Although androgen deprivation therapy is an effective treatment against recurrent disease, most of these patients eventually develop androgen-refractory prostate cancer, which is insensitive to conventional treatments. Therefore, more effective and less toxic therapies are urgently needed. Immunotherapy has been shown to be a promising treatment for prostate cancer, especially in patients with metastatic castration-resistant prostate cancer [2-4]. Manipulating the immune system to eliminate malignant cells is a promising treatment for cancer, but until recently It has also had sporadic clinical success [4-6]. The recent Food and Drug Administration (FDA) approval of immunotherapy-based vaccines/drugs sipuleucel-T (Provenge) and ipilimumab (Yervoy) represents a milestone in the field of cancer immunotherapy [7,8]. In addition, the phase III clinical trial of gp100 peptide in melanoma also produced very encouraging clinical results [9]. However, the reported clinical benefits of these agents fall far short of complete response and permanent cure. In the case of sipuleucel-T, patients had a survival benefit of only 4.1 months, with no objective tumor regression or substantial changes in prostate-specific antigen (PSA) levels. Recent studies using animal models have further revealed the importance of tumor-specific antigens in inducing immune responses against developing tumors [10], which has prompted additional efforts to identify such antigens for cancer immunotherapy. Furthermore, since some major rejection antigens may be lost or altered by T cell selection and killing [11], the best strategy is to target multiple tumor antigens present on individual tumors for immunotherapy.

So far, a variety of prostate-specific tumor antigens have been identified, including PSA[12,13], prostein[14,15], prostate stem cell antigen (PSCA)[16], prostate-specific membrane antigen (PSMA)[17-19 ], prostatic acid phosphatase (PAP) [20], and transient receptor potential p8 (trp-p8) [21]. Furthermore, HLA class I-restricted epitopes derived from these tumor antigens have been described [22]. A disadvantage of single tumor antigen-based immunotherapy is the potential for immune escape. Therefore, there is a need to identify additional prostate cancer-specific antigens to develop more effective and antigen-specific vaccines for patients with metastatic prostate cancer.

Contents of the invention

The prostate-specific G protein-coupled receptor (PSGR) is a member of the G protein-coupled olfactory receptor family and is highly expressed in prostate cancer cells compared to normal prostate cells [23-25], suggesting that PSGR can be targeted to develop anti- New immunotherapy strategies for prostate cancer.

We determined that PSGR is recognized by T cells and characterized the PSGR-derived T-cell epitopes used for T-cell recognition. Twenty-one peptides predicted to bind to HLA-A2 molecules were selected and synthesized, and their stimulation of T in PBMCs from healthy subjects and prostate cancer patients was evaluated in vitro based on interferon gamma (IFN-γ) release measured by ELISA or ELISPOT assay. cell capacity. Three peptides, PSGR3, PSGR4 and PSGR14 (see below and Table 1) were found to induce IFN-γ release in peripheral T cells from healthy subjects and prostate patients. Importantly, these peptide-specific T cells recognized HLA-A2 ⁺ , PSGR-expressing LNCaP cells in an HLA class I-dependent manner.

Therefore, in one embodiment, the present invention provides a composition, comprising a polypeptide composed of the amino acid sequence of PSGR3, PSGR4 or PSGR14, or a combination of two or more of these three peptides, and a pharmaceutically acceptable carrier.

In one aspect, the present disclosure provides a method of treating or preventing prostate cancer comprising administering to a patient in need thereof an effective amount of a composition of the present invention.

In an embodiment, the present disclosure provides a method of treating prostate cancer in a human patient comprising the step of administering to the patient an amount of a composition of the present disclosure effective to stabilize or reduce serum prostate specific antigen (PSA), particularly PSGR levels. In certain aspects, the methods of the present disclosure further comprise administering granulocyte macrophage colony stimulating factor (GM-CSF). In some aspects, the composition and GM-CSF are co-administered, and in further embodiments the composition and GM-CSF are administered simultaneously, and in still further embodiments the composition and GM-CSF are administered Apply sequentially.

In certain embodiments, PSGR is administered as a composition of PSGR peptide-loaded dendritic cells, eg, in multiple injections.

Administration of the compositions or vaccines of the present disclosure may in various aspects be intradermal. Therefore, the present disclosure also provides a vaccine, comprising: (i) a polypeptide composed of the amino acid sequence of PSGR3, PSGR4 or PSGR14, or a combination of two or more of these three peptides, and (ii) a pharmaceutically acceptable carrier. In certain aspects, the vaccine further comprises granulocyte macrophage colony stimulating factor (GM-CSF). In a further aspect, the vaccine further comprises a toll-like receptor 9 (TLR9) agonist in an amount effective to increase the T cell immune response. In a specific aspect, the TLR9 agonist is a CpG-oligodeoxynucleotide (CpG-ODN).

In a further embodiment, the vaccine further comprises a CTLA4 inhibitor in an amount effective to increase the T cell immune response, and in specific aspects the CTLA4 inhibitor is a monoclonal antibody.

In additional embodiments, the vaccine further comprises a PD-1 inhibitor in an amount effective to increase the T cell immune response. In a specific aspect the PD-1 inhibitor is a monoclonal antibody.

The present disclosure also provides methods of vaccinating an individual comprising administering to the individual an amount of a vaccine of the invention effective to vaccinate the individual. In certain aspects, the vaccines of the invention are co-administered with GM-CSF, and in further aspects are co-administered in multiple injections. In a further aspect, the PSGR peptide is administered concurrently with GM-CSF, and in yet a further aspect it is administered sequentially with GM-CSF.

Description of drawings

Figure 1 shows that PSGR-derived peptides induce peptide-specific T cells. Expanded PSGR peptide-specific T cells were tested for recognition by ELISA assay against T2 cells preloaded with titrated concentrations of peptide (0-20 μg/ml) (A). Expanded PSGR3T cells (B and E), PSGR4T cells (C and F) and PSGR14T cells (D and G) were incubated with T2 cells alone ( ^1x104 cells/well) in complete medium (CM), or with T2 cells preloaded with the corresponding peptide (5 μg/mL) or a control peptide as a negative control were co-incubated. Cells were incubated for 18-24 hours and IFN-γ secretion in the supernatant was measured by ELISA assay (B, C and D). IFN-γ spot forming cells (SFC) were counted by ELISPOT assay (E, F and G). Data are plotted as mean ± SD. Results are representative of at least three independent experiments. *P<0.05, **P<0.01, ***P<0.001 versus control (T2 cells alone or T2 cells loaded with control peptide).

Figure 2 shows that T cells specific for PSGR-derived peptides recognize HLA-A2 positive PSGR expressing LNCaP prostate cancer cells. Expression of PSGR mRNA in different cell lines was determined by RT-PCR (A). PSGR-derived peptide-specific T cells were tested for cytotoxicity against PC3 and LNCaP by LDH assay (B). Data from B are plotted as mean ± SD. Results are representative of at least three independent experiments. *P<0.05 vs. control.

Figure 3 shows that T cell responses induced by PSGR-derived peptides are CD8 ⁺ T cell dependent and HLA-I restricted. PSGR-derived peptide-specific T cells were incubated with T2 cells loaded with or without the given peptide in the presence of GolgiStop in 48-well plates at 37°C for 4 hours. Cells were stained with anti-CD8 and anti-IFN-γ and then analyzed on a FACScalibur instrument (A). PSGR-derived peptide-specific T cells were inoculated with LNCaP cells in culture alone, or with LNCaP in the presence of anti-HLA-I mAb (W6/32), HLA-II mAb or control mAb (anti-CD19 mAb). Cells were co-incubated. After 4 hours of incubation, cytotoxicity to LNCaP was determined by LDH assay (B). Data from B are plotted as mean ± SD. Results are representative of at least three independent experiments. *P<0.05 vs. control.

specific embodiment

It has been established that CD8 ⁺ T cells play a key role in tumor development and progression. Peptide epitopes derived from tumor-associated antigens (TAAs) can be recognized as antigens by T cells in the presence of MHC-I molecules [32,33]. The identification of TAAs and their peptides recognized by T cells is crucial for the development of effective cancer vaccines.

The goal of this study was to identify HLA-A2-binding PSGR-derived epitopes recognized by CD8 ⁺ T cells in PBMCs from healthy subjects and prostate cancer patients. PSGR protein sequences were scanned for HLA-A2 binding peptides based on HLA-A2 binding motifs using three different computer prediction algorithms including BIMAS, SYFPEITHI, and Rankpep. Only peptides that were successfully predicted by at least 2 of 3 different in silico prediction algorithms were included. According to this criterion, 21 kinds of 9 amino acid or 10 amino acid peptides were selected in this study. All these peptides were tested for their ability to stimulate the release of IFN-γ from PBMCs from healthy subjects or prostate cancer patients. Of the 21 peptides, 3 peptides frequently induced peptide-specific T cell responses in PBMCs obtained from healthy subjects or cancer patients, and these peptide-specific T cells also recognized HLA-A2 ⁺ , PSGR-expressing LNCaP cells, suggesting that these Peptides are naturally processed by prostate cancer cells.

PSGR is a prostate tissue-specific gene that has homology to the G protein-coupled olfactory receptor gene family and is specifically expressed in human prostate tissue [23-25]. The expression of PSGR in human prostate intraepithelial neoplasia and prostate tumors was significantly higher than that in normal tissues [25]. Interestingly, although PSGR has been recognized as a novel target for prostate cancer immunotherapy, PSGR-derived T-cell epitopes have not yet been identified. To our knowledge, this is the first report to identify and characterize a PSGR-derived epitope recognized by CD8 ⁺ T cells. The identification of PSGR-derived epitopes recognized by T cells further validates PSGR as a promising target for the development of cancer vaccines.

Most TAAs are self-antigens [34], thus, self-tolerance may occur in an attempt to protect an individual from the development of autoimmunity. This is considered a major obstacle to the induction of TAA-specific T cells capable of eradicating tumors in vivo. However, in our study, although PSGR was expressed in normal prostate tissues, since T cell responses to PSGR-derived epitopes were frequently detected in PBMCs from healthy subjects or prostate cancer patients, immune tolerance to PSGR could destroyed.

Numerous immunization-based immunotherapy clinical trials have been conducted using tumor lysates, TAA proteins, TAA peptides, and RNA or DNA encoding TAAs. However, most of these trials did not yield the desired results. One reason is that the expression of these TAAs is heterogeneous in tumors from different patients and can also vary between metastases obtained from one patient [35,36], so when immunotherapeutic approaches are based on only one TAA Immune escape occurs. To avoid immune escape, vaccine-based immunotherapeutic strategies targeting multiple different tumor antigens are crucial for the development of successful cancer vaccines. Therefore, although PSA[12,13], PSCA[16], PSMA[17-19], PAP[20], Prostein[14,15], trp-p8[21] have been identified in recent years Many prostate-specific tumor antigens have been identified, and additional prostate-specific tumor antigens for T cell-based immunotherapy still need to be identified.

Based on phase III studies, the FDA recently approved a cancer vaccine, Sipuleucel-T, for the treatment of patients with advanced prostate cancer [8]. Sipuleucel-T is prepared from autologous PBMCs containing antigen-presenting cells, and the autologous PBMCs are incubated with a recombinant protein consisting of PAP linked to macrophage colony-stimulating factor (GM-CSF). It is speculated that Sipuleucel-T works in part by increasing PAP-specific CD8 ⁺ T cell responses, which further confirms the importance of tumor antigen-specific CD8 ⁺ T cells induced by cancer vaccines. So far, Sipuleucel-T is the first cellular immunotherapy agent approved by the FDA for the treatment of cancer patients. The FDA approval of Sipuleucel-T as a therapeutic cancer vaccine not only verified the efficacy of cancer immunotherapy, but also provided a strong impetus for the field of cancer immunology [37]. Therefore, the identification and development of more novel TAAs recognized by CTLs, including PSGR and peptide derivatives, is absolutely critical to facilitate the development of future effective cancer vaccines against prostate cancer as well as other types of cancer.

In addition, epitopes recognized by CD8 ⁺ T cells can be used as a diagnostic tool to monitor peptide-specific CD8 ⁺ T cells in individuals during immunotherapy, thereby identifying the optimal time period of immunotherapy during treatment, including anti-tumor immunity. Whether an individual requires subsequent immunotherapy when power declines.

In conclusion, we have identified 3 novel PSGR-derived CTL epitopes. Since PSGR expression is strongly upregulated in human prostate cancer, PSGR-derived peptides could be used as diagnostic tools, or immunotherapeutic targets for anticancer vaccines alone or in combination with other epitopes derived from other prostate-specific antigens.

The invention is illustrated by the following examples, which are not intended to be limiting in any way.

Example

Materials and Methods

Healthy donors and prostate cancer patients

Ten HLA-A2 ⁺ prostate patients and 10 HLA-A2 ⁺ healthy subjects participated in this study after obtaining written informed consent. All protocols were approved by the Institutional Review Board (IRB) of Baylor College of Medicine prior to initiation of the study. 20 ml of peripheral blood were obtained from each individual and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep (Nycomed Pharma AS; Oslo, Norway). Freshly isolated PBMCs were frozen in 1 ml freezing medium containing 90% FCS and 10% dimethylsulfoxide (DMSO) at -140°C for subsequent use. HLA-A2 molecule expression on PBMCs obtained from cancer patients and healthy subjects was verified by flow cytometry with FITC-labeled HLA-A2 mAb BB7.2 (BDPharmingen, San Diego, CA, USA).

cell line

T2 cells (HLA-A2 ⁺ TAP-deficient cell line), PC3 cells (HLA-A2-negative prostate cancer cell line), and LNCaP cells (HLA-A2-positive prostate cancer cell line) were purchased from American Type Culture Collection ) (ATCC; Manassas, VA, USA). All cell lines were maintained in RPMI-1640 medium (Mediatech; Manassas, VA, USA) supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin and streptomycin.

peptide

Based on HLA-A2 binding motifs using BIMAS (http://www-bimas.cit.nih.gov/molbio/hla_bind/), SYFPEITHI (http://www.syfpeithi.de/), and Rankpep (http:/ /bio.dfci.harvard.edu/Tools/rankpep.html) predicted 21 PSGR-derived peptides (Table 1). Only epitopes predicted by at least 2 of these algorithms were selected for further testing. Peptides were synthesized by a solid-phase method using a peptide synthesizer (AApptec, Inc.; Louisville, KY, USA), purified by reverse-phase high-performance liquid chromatography and verified by mass spectrometry. Synthesized peptides were dissolved in DMSO at a concentration of 10 mg/mL and stored at -80°C until further use.

In vitro stimulation of peptide-specific T cells in PBMCs

PBMCs ( ^1x105 cells/well) from healthy subjects or patients with prostate cancer were incubated with standard peptide concentrations of 20 μg/mL for each peptide in 200 μL T cell culture medium (TCM) on 96-well U-bottom plates (BD, Franklin Lakes, NJ, USA). ), T cell medium consisted of RPMI1640 (Mediatech, Manassas, VA, USA), 10% human AB serum (Valley Biomedical, Winchester, USA), 50 μM 2-mercaptoethanol, 100 U/mL interleukin-2 ( IL-2), and 0.1mMMEM non-essential amino acid solution (Invitrogen, Grand Island, NY, USA). Half of the TCM was removed every 5 days and replaced with fresh TCM containing peptide (20 μg/mL). After 14 days of culture, cells were harvested and tested for their ability to produce IFN-γ in response to T2 cells (1×10 ⁴ cells/well) pre-loaded with PSGR peptide (5 μg/mL) or a control peptide (irrelevant HLA - A2 binding peptide: NLLTHVESL). After 18 hours of incubation, supernatants were collected and assayed for IFN-γ release by ELISA.

Table 1. Predicted HLA-A2-binding peptides derived from prostate-specific G-protein-coupled receptors (PSGRs)

Rapid Expansion Protocol (REP) for PSGR Peptide-Specific T Cells

PSGR peptide-specific T cells were expanded by the Rapid Expansion Protocol (REP) as previously described [29] with minor modifications. Briefly, 20 mL RPMI-1640 supplemented with 10% human AB serum, 50 μM 2-mercaptoethanol, and 30 ng/mL OKT antibody (OrthoBiotech, Bridgewater, NJ) was irradiated with 20×10 ⁶ isotypes on day 0 in T25 flasks. Allogeneic PBMCs were co-cultured with 5x10 ⁶ irradiated Epstein-Barr virus (EBV) for 0.1-0.5x10 ⁶ PSGR peptide-specific T cells. Flasks were incubated upright at 37 °C in 5% _CO2 . IL-2 (300 IU/mL) was added on day 1 and day 5, half of the cell culture supernatant was removed and supplemented with fresh medium containing 300 IU/mL IL-2. 14 days after the start of REP, the cells were harvested and frozen for future experiments.

ELISA test

Cytokine release was measured by overnight coating of 96-well ELISA plates (ThermoFisher Scientific; Rochester, NY, USA) with 1 μg/mL anti-human IFN-γ (Pierce Biotechnology; Rockford, IL, USA). Plates were washed 6 times with PBS containing 0.05% Tween-20 (wash solution) to remove unbound coated antibody, and blocked with 1% BSA/PBS for 2 hours at room temperature. Afterwards, 50 μL of supernatant was added to each well and incubated at room temperature for 1 hour, followed by the addition of 50 μL of 0.5 μg/mL biotinylated anti-human IFN-γ (Pierce Biotechnology; Rockford, IL, USA), and the plate was incubated at room temperature. Incubate for another 1 hour. After incubation, plates were washed and incubated for 30 minutes with poly-HRP streptomycin (ThermoFisher Scientific; Rochester, NY, USA) diluted 1:5000 in PBS/1% BSA. Plates were washed and 100 μL of TMB substrate solution (Sigma-Aldrich Co.; St. Louis, MO, USA) was added to each well. _The chromogenic reaction was stopped with _2NH2SO4 and the plate was read at 450nm using an ELISA plate reader.

IFN-γELISPOT detection

After in vitro expansion, IFN-γ ELISPOT assay was performed to quantify peptide-specific cytotoxic T lymphocytes (CTLs) as previously described [27]. Briefly, 96-well ELISPOT plates (Millipore; Bedford, MA, USA) were coated overnight at 4°C with 7.5 μg/mL anti-human IFN-γ (Pierce Biotechnology; Rockford, IL, USA). Plates were washed 6 times with sterile PBS to remove unbound coating antibody. T cells were seeded at 1x105 cells per well and incubated with T2 cells alone, T2 cells loaded with PSGR peptide ( ⁵ μg/mL) or an irrelevant peptide as a negative control. Cells stimulated with 5 μg/mL OKT3 antibody (OrthoBiotech; Bridgewater, NJ, USA) were used as a positive control. After incubating the samples for 18-20 hours at 37°C in 5% CO ₂ , the plates were washed with washing solution. 0.75 μg/mL biotinylated anti-human IFN-γ (Pierce Biotechnology; Rockford, IL, USA) was added and the plate was incubated at room temperature for 2 hours. After incubation, plates were washed with wash solution and further incubated for 1 hour with poly-HRP streptomycin (ThermoFisher Scientific; Rochester, NY, USA) diluted 1:1000 in PBS/1% BSA. Plates were washed and 200 μL of 4-chloro-1-naphthol substrate (Sigma-Aldrich Co.; St. Louis, MO, USA) was added to each well. Finally, the plates were washed under running water and dried at room temperature. IFN-γ spot forming cells (SFCs) were counted using an ELISPOT reader (CTL Technologies, Minneapolis, MN, USA).

RNA extraction and RT-PCR

RNA extraction and RT-PCR were performed as previously reported [30]. Briefly, total RNA was extracted from prostate cancer cells with 1 mL of Trizol reagent (Invitrogen; Carlsbad, CA, USA). 3 micrograms of RNA were reverse transcribed into cDNA in a volume of 30 μl, and 1 μl of each cDNA was used for subsequent PCR reactions with PSGR-specific primer pairs: Primer 1: 5'-GAAGATCTATGAGTTCCTGCAACTTC-3' (SEQ ID NO:22), Primer 2: 5'-CCCAAGCTTTCACTTGCCTCCCACAG-3' (SEQ ID NO: 23). β-actin was used as a loading control: Primer 1: 5'-CATGATGGAGTTGAAGGTAGTTTCG-3' (SEQ ID NO: 24); Primer 2: 5'-CAGACTATGCTGTCCCTGTACGC-3' (SEQ ID NO: 25). The PCR reaction was performed under the following conditions: 94 °C for 2 min, 94 °C for 30 sec, 56 °C for 30 sec, 72 °C for 1 min 20 sec, 35 cycles in total, 72 °C for 10 min, and β-actin for 25 cycles . An equal amount of PCR product was then loaded and detected by gel electrophoresis.

Cytotoxicity Assay

Cytotoxicity of PSGR-derived peptide-specific T cells against PC3 and LNCaP was tested by lactate dehydrogenase (LDH) assay (Promega; Madison, WI, USA). Assays were performed according to the manufacturer's instructions. LDH release was calculated based on the following formula:

Cytotoxicity (%) = (experiment - effector spontaneous - target spontaneous LDH release) / (target maximum - target spontaneous LDH release) x 100

Spontaneous release was determined using target cells alone or effector cell supernatants alone, and maximal release was determined using target cell supernatants incubated with the lysis solution included in the LDH kit. To determine whether T cell recognition is HLA-I restricted, anti-HLA-I, anti-HLA-II, or anti-CD19 mAbs (all from ATCC, Manassas, VA, USA) were added to the wells at the beginning of the culture.

Intracellular IFN-γ cytokine staining

PSGR-derived peptide-specific T cells (0.5-1×10 ⁶ ) were incubated with 0.5×10 ⁶ T2 cells loaded with or without peptide (5 μg/mL) in the presence of GolgiStop (BDPharmingen, San Diego, CA, USA) on 48-well plates for 37 Cultivate for 4 hours. Cells were stained with anti-CD8 and anti-IFN-γ and analyzed using a FACScalibur instrument.

statistics

Quantitative differences between experimental wells and controls in ELISA and ELISPOT assays were analyzed using Student's t-test. P<0.05 was considered significant.

result

Induction of PSGR-derived peptide-specific CTLs in healthy donors

To determine whether there are PSGR-responsive T-cell precursors in healthy subjects, we obtained PBMCs from 10 HLA-A2+ healthy donors and treated them in vitro with 21 PSGR-derived peptides containing HLA-A2 binding motifs (Table 1 ) each stimulates it. After 2 weeks of peptide stimulation, supernatants from peptide-stimulated T cells were analyzed by ELISA assay to detect IFN-γ release in response to T2 cells loaded with or without the corresponding peptide. As shown in Table 2, 13 PSGR-derived peptides were able to induce peptide-specific T cell responses in at least 1 in 10 healthy subjects. Importantly, PSGR3, PSGR4 and PSGR14 could induce T cell responses in 7 out of 10 healthy subjects, suggesting that these 3 peptides are immunogenic and potentially able to expand antigen-specific T cells in healthy subjects.

Table 2. Peptide-specific T cell induction in PBMCs of 10 HLA-A2 ⁺ healthy subjects

Note: Values represent IFN-γ concentration (pg/ml) in the supernatant; - not completed

Presence of PSGR-derived peptide-specific CTLs in prostate cancer patients

Since peptide-specific T cells against PSGR3, PSGR4 and PSGR14 were found in more than 70% of healthy subjects, we speculated that CTL precursors recognizing these 3 peptides might also be high in PBMCs of prostate cancer patients. To test our hypothesis, we examined whether these 3 peptide candidates could induce peptide-specific CTLs from HLA-A2 ⁺ prostate cancer patient PBMCs. PBMCs from prostate cancer patients were collected and stimulated in vitro with PSGR3, PSGR4 or PSGR14 peptides. As shown in Figure 3, PSGR3, PSGR4 and PSGR14 did induce peptide-specific CTLs from prostate cancer patient PBMCs.

Table 3. Peptide-specific T cell induction in PBMCs of HLA-A2 ⁺ prostate cancer patients

Note: the value represents the concentration of IFN-γ in the supernatant (pg/ml)

PSGR-derived peptides specifically recognize prostate cancer cell lines

To obtain a large number of PSGR peptide-specific T cells for further analysis, we expanded the PSGR peptide-specific T cells identified in Tables 2 and 3. To determine the effective peptide concentration of loaded T2 cells recognized by T cells, we performed peptide titration experiments. As shown in Figure 1A, for all three peptides, a peptide concentration of 5 μg/ml was sufficient to saturate the HLA-A2 molecule binding sites on T2 cells recognized by T cells. Therefore, we have always used this peptide concentration to preload T2 cells in ELISA and/or ELISPOT assays. Expanded T cells retained antigen specificity and secreted significant amounts of IFN-γ upon stimulation with T2 cells loaded with the corresponding peptides, but not control peptides (Fig. 1A, B, C, D). ELISPOT assay further confirmed the presence of PSGR peptide-specific T cells among the expanded T cells (Fig. 1E, F, G).

To determine whether PSGR-derived peptide-specific T cells could recognize and kill HLA-A2 ⁺ PSGR-expressing prostate cancer cells, we used the HLA-A2-negative PC3 cell line and the HLA-A2-positive LNCaP prostate cancer cell line. PSGR expression in these two cell lines was detected by RT-PCR. Consistent with a previous report [31], PSGR was highly expressed in LNCaP but not in PC3, DU145 or the normal prostate cell line PNT1A (Fig. 2A). As shown in Figure 2B, PSGR3, PSGR4, or PSGR14-specific T cells from healthy donors and patients could recognize and kill HLA-A2-positive, PSGR-expressing LNCaP, but not HLA-A2-negative PC3 cells. These results suggest that PSGR peptide-specific T cells recognize T cell epitopes that are internally processed and displayed by prostate tumor cells.

T cells recognize PSGR-derived peptides in an HLA-I-restricted manner

To test whether the responses induced by PSGR-derived peptides were dependent on CD8 ⁺ T cells, we co-cultured PSGR-derived peptide-specific T cells with T2 cells loaded with or without the corresponding peptides in the presence of GolgiStop3 in 48-well plates for 4 hours. CD8 molecular and intracellular IFN-γ staining was then performed. Only CD8 ⁺ T cells were found to produce IFN-γ in response to T2 cells loaded with the corresponding peptide, whereas CD4 ⁺ T cells did not produce IFN-γ (data not shown).

To determine whether the recognition of LNCaP cells by PSGR-derived peptide-specific T cells was HLA-I restricted, we compared LNCaP cells with PSGR-derived peptide-specific T cells in the presence of anti-HLA-I mAb (W6/32) or a control antibody. (HLA-II monoclonal antibody or anti-CD19 monoclonal antibody) in the presence of co-cultivation. As shown in Figure 3B, the cytotoxicity of these peptide-specific T cells was completely inhibited by the addition of anti-HLA-I mAb, but not by anti-HLA-II (HLA-DR) or control mAb (anti-CD19) was inhibited, suggesting that recognition of LNCaP cells by PSGR-derived peptide-specific T cells is HLA-I-restricted.

Although the invention has been described in terms of specific embodiments, it is to be understood that changes and modifications will occur to those skilled in the art. Accordingly, no limitations other than those in the claims should be placed on the invention.

All documents cited in this application are hereby incorporated by reference in their entirety for the disclosures they describe.

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Claims (20)

CLAIMS 1. A method of treating prostate cancer comprising administering to a patient in need thereof an effective amount of a peptide selected from PSGR3, PSGR4 and PSGR4, or a combination of two or more of these three peptides. 2. The method of claim 1, wherein the peptides are co-administered. 3. The method of claim 1, wherein the peptides are administered simultaneously. 4. The method of claim 1, wherein the peptides are administered sequentially. 5. The method of claim 1, wherein the route of administration is intradermal. 6. The method of claim 1, further comprising administering to the patient granulocyte macrophage colony stimulating factor (GM-CSF). 7. The method of claim 6, wherein the peptide and GM-CSF are co-administered in multiple injections. 8. The method of claim 6, wherein the peptide and GM-CSF are administered simultaneously. 9. The method of claim 6, wherein the peptide and GM-CSF are administered sequentially. 10. The method of claim 1 or claim 6, further comprising administering to the patient a TLR9 agonist, CTLA4 inhibitor or PD-1 inhibitor in an amount effective to increase the T cell immune response. 11. The method of claim 10, wherein the TLR9 agonist is a CpG-oligodeoxynucleotide (CpG-ODN). 12. The method of claim 10, wherein the CTLA4 inhibitor is a monoclonal antibody. 13. The method of claim 10, wherein the PD-1 inhibitor is a monoclonal antibody. 14. The method of claim 11, wherein the peptide is administered at 1, 4, and 10 weeks, and thereafter every 6 months for up to 4 years. 15. The method of claim 10, wherein the peptide is administered at 1, 4 and 10 weeks, and then administered every 6 months for up to 4 years, and wherein the CTLA4 inhibitor is administered at 1, 4 and 10 weeks, and subsequently Administered every 8 weeks for up to 52 weeks. 16. The method of claim 10, wherein the peptide is administered at 1, 4, and 10 weeks, and is subsequently administered every 6 months for up to 4 years, and wherein the PD-1 inhibitor is administered at 1, 4, and 10 weeks, and subsequently administered every 8 weeks for up to 52 weeks. 17. A composition comprising: (i) a pharmaceutically acceptable carrier, (ii) one or more PSGR peptides selected from the group consisting of PSGR3, PSGR4 and PSGR14. 18. The composition of claim 17, wherein the composition is formulated as a vaccine. 19. A kit comprising the composition of claim 17, instructions for administering the composition, and a device for administering the composition to a patient. 20. A method of preventing prostate cancer in a human subject in need thereof, the method comprising administering the composition of claim 18 to the human subject.