不同类型肿瘤抗原致敏的树突状细胞体外诱导前列腺癌特异性CTL的研究

目的:研究肿瘤冻融抗原及弱酸洗脱提取的肿瘤抗原肽分别冲击致敏树突状细胞(dendritic cells,DCs)体外诱导前列腺癌特异性细胞毒性T淋巴细胞(CTL)的效应。方法:采用反复冻融法和弱酸洗脱法,分别获取前列腺癌细胞株PC-3的肿瘤抗原,应用重组人GM-CSF、IL-4体外培养诱导外周血单个核细胞获得DC,并分别负载两种前列腺癌肿瘤抗原,检测其体外激活肿瘤特异性CTL的能力,用LDH释放法检测其对PC-3的体外杀伤效应。结果:用枸橼酸-磷酸盐缓冲液洗脱或反复冻融PC-3(2×107)后获得的肿瘤抗原蛋白含量分别为(312.2±7.9)μg和(963.0±25.3)μg,负载两种前列腺癌细胞抗原的DCs致敏的CTL对PC-3细胞有明显的杀伤作用,而且负载弱酸洗脱提取的肿瘤抗原肽的DCs致敏的特异性CTL具有更明显的杀伤作用[(60.4±5.52)%vs(43.7±4.11)%,P<0.01)]。结论:弱酸洗脱和反复冻融方法都可以有效获取前列腺癌抗原,用于体外致敏DCs后,均具有活化CTL的能力,而且弱酸洗脱提取的肿瘤抗原肽更为有效。     

Identification of HLA-DRB1*1501-restricted T-cell epitopes from human prostatic acid phosphatase

BACKGROUND: The crucial role of CD4 T-cells in anti-tumor immune response is widely recognized, yet the identification of HLA class II-restricted epitopes derived from tumor antigens has lagged behind compared to class I epitopes. This is particularly true for prostate cancer. Based on the hypothesis that successful cancer immunotherapy will likely resemble autoimmunity, we searched for the CD4 T-cell epitopes derived from prostatic proteins that are restricted by human leukocyte antigen (HLA)-DRB1*1501, an allele associated with granulomatous prostatitis (GP), a disease that may have an autoimmune etiology. One of the antigens implicated in the development of autoimmunity in the prostate is prostatic acid phosphatase (PAP), which is also considered a promising target for prostate cancer immunotherapy. METHODS: We immunized transgenic (tg) mice engineered to express HLA-DRB1*1501 with human PAP. A library of overlapping 20-mer peptides spanning the entire human PAP sequence was screened in vitro for T-cell recognition by proliferative and interferon (IFN)-gamma enzyme-linked immunosorbent spot (ELISPOT) assays. RESULTS: We identified two 20-mer peptides, PAP (133-152), and PAP (173-192), that were immunogenic and naturally processed from whole PAP in HLA-DRB1*1501 tg mice. These peptides were also capable of stimulating CD4 T lymphocytes from HLA-DRB1*1501-positive patients with GP and normal donors. CONCLUSIONS: These peptides can be used for the design of a new generation of peptide-based vaccines against prostate cancer. The study can also be helpful in understanding the role of autoimmunity in the development of some forms of chronic prostatitis.     

Identification of an immunodominant H-2D(b)-restricted CTL epitope of human PSA

BACKGROUND: Human prostate specific antigen (PSA) is expressed selectively in prostate epithelium and is a potential target for the immunotherapy against prostate cancer. Various PSA-based vaccines have been reported to induce cytotoxic T lymphocyte (CTL) responses in animal models. Here, we present the identification and validation of an immunodominant CTL epitope of PSA in C57Bl/6 mice (H-2(b)). METHODS: PSA-specific CTLs were induced by immunization with a plasmid expressing PSA. Epitope specificity of the CTLs was determined by their reactivity against a panel of C-terminus truncated or mutated PSA proteins and use of bioinformatical prediction with the SYFPEITHI algorithm. RESULTS: The majority of PSA-specific CTLs were directed against a single H-2D(b) restricted epitope corresponding to the amino acid residues 65-74 (HCIRNKSVIL) of the protein. The CTLs had similar functional avidity against two putative H-2D(b) binding peptides: a 9-aa-long psa65-73 (HCIRNKSVI) and a 10-aa-long psa65-74 (HCIRNKSVIL). CONCLUSIONS: We demonstrate that the psa65-73 peptide can be used for reactivation of PSA-specific CTLs in vitro and ex vivo, and H-2D(b) pentamers assembled with this peptide are an efficient tool for monitoring of PSA-specific CTL responses after DNA vaccination.     

In vitro propagated dendritic cells from prostate cancer patients as a component of prostate cancer immunotherapy

T cell-mediated cancer immunotherapy requires efficient antigen-presenting cells. Dendritic cells (DCs) are arguably the most efficient antigen-presenting cells studied to date. Individuals with prostate cancer often undergo various therapies which may compromise their immune system, including the state of their DC precursors. We report the in vitro propagation of DCs from peripheral blood of patients with prostate cancer, most of whom are in clinical stages D₁ or D₂ and have undergone radiation therapy. After 7 days in culture, the number of DCs recovered were 20-50-fold higher than those isolated directly from peripheral blood. This number is comparable to findings of previous studies with healthy individuals. Cultured patients' DCs were capable of presenting tetanus toxoid to autologous T cells in vitro. Furthermore, T cells from 2 of 4 patients proliferated when cultured with their DCs and the lysate of a human prostate cancer cell line (LNCaP), demonstrating the potential role of autologous DCs in prostate cancer immunotherapy studies. © 1995 Wiley-Liss, Inc.     

Clonal T‐cell response against autologous pleomorphic malignant fibrous histiocytoma antigen presented by retrieved HLA‐A*0206

Towards the goal of identifying tumor‐rejection antigens on eradicated tumors in bone and soft tissue sarcomas, we evaluated the immune response against antigens presented by lost HLA class I molecules. Tumor specimens and peripheral blood samples were obtained from a 70‐year‐old woman with pleomorphic malignant fibrous histiocytoma. Over 1‐year culture, a tumor cell line (MFH2004) was established. A B‐cell line infected with Epstein–Barr virus (B2004‐EBV) was developed from the blood samples. HLA genotypes of B2004‐EBVcells were A*0206/2402, B*4006/4601, and C*0102/0801, whereas MFH2004 cells were defective for A*0206, B*4006, and C*0102. Loss of HLA‐A2 expression was also proved immunohistochemically in the primary tumor tissues. Lost HLA‐A2 in MFH2004 cells was retrieved by transfection of HLA‐A*0206 cDNA to develop MFH2004‐A2. Attempts to induce CTLs by mixed culture with autologous T cells and MFH2004 cells resulted in failure. In contrast, those with MFH2004‐A2 induced CTL clones CTL2004‐c6 and CTL2004‐c17. These CTL clones specifically killed MFH2004‐A2 but not MFH2004 or B2004‐EBV in an HLA‐A2‐restricted manner. These findings suggest that CTL2004‐c6 and CTL2004‐c17 recognize autologous tumor‐rejection antigens presented by HLA‐A*0206, which may have been expressed by tumor cells that had been eradicated by the host's immunosurveillance system. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:271–278, 2008     

Clinical development of immunotherapy for prostate cancer

Prostate cancer is the most common cancer in men, and the second leading cause of cancer‐related death in Western countries. Prostate cancer‐related death occurs in patients with metastatic castration‐resistant prostate cancer. Although several new drugs for castration‐resistant prostate cancer have been approved, each of these has prolonged survival by just a few months. Consequently, new therapies are sorely needed. Recently, it has been recognized that immunotherapy is an effective treatment for prostate cancer patients. Several strategies, such as cancer vaccines and immune checkpoint inhibitors, have been investigated in clinical studies for prostate cancer patients. In the present review, the results of the most recent clinical studies investigating immunotherapy in prostate cancer patients are reported, and the future clinical development of immunotherapy for prostate cancer is discussed.     

Identification of polycomb group protein enhancer of zeste homolog 2 (EZH2)-derived peptides immunogenic in HLA-A24+ prostate cancer patients

BACKGROUND: Antigens overexpressed in metastatic prostate cancer are appropriate targets in anti-cancer immunotherapy, and one candidate is the polycomb group protein enhancer of zeste homolog 2 (EZH2). METHODS: Eleven EZH2-derived peptides were prepared based on the HLA-A24 binding motif. These peptide candidates were screened first by their ability to be recognized by immunoglobulin G (IgG), and then by their ability to induce peptide-specific cytotoxic T lymphocytes (CTLs). RESULTS: IgGs reactive to three EZH2 peptides (EZH2-243 to -252, EZH2-291 to -299, and EZH2-735 to -;742) were detected in the plasma of almost half of prostate cancer patients. Among them, the EZH2-291 to -299 and EZH2-735 to -742 peptides effectively induced HLA-A24-restricted and prostate cancer-reactive CTLs from prostate cancer patients. The cytotoxicity was mainly dependent on EZH2 peptide-specific and CD8+ T cells. CONCLUSIONS: These EZH2-291 to -299 and EZH2-735 to -742 peptides could be promising candidates for peptide-based immunotherapy for HLA-A24+ prostate cancer patients with metastases.     

应用MAGE-3抗原肽诱导肝癌患者细胞毒T淋巴细胞反应的研究

目的研究MAGE 3抗原肽对肝细胞癌 (HCC)患者进行免疫治疗的可行性。方法微量细胞毒法检测HCC患者中HLA A2表达情况 ,RT PCR方法检测肝癌组织MAGE 3基因mRNA表达情况。用免疫磁珠从患者外周血单个核细胞 (PBMC)中分离出CD8阳性细胞作为效应细胞 ;将其余自体CD8阴性的PBMC与MAGE 3抗原肽 (FLWGPRALV)短暂孵育 ,经γ射线照射后作为抗原呈递细胞 (APC) ,与CD8阳性效应细胞进行混合淋巴细胞培养 ,作为实验组 ,7d后再加入APC刺激患者效应细胞 1次。每周计数效应细胞数量 ,培养第 15天时作为细胞毒T淋巴细胞 (CTL)检测其杀伤活性。以表达MAGE 3和HLA A2的肝癌细胞系HLE作为靶细胞 ,采用IFN γ细胞因子分泌法 ,通过流式细胞仪检测具有分泌γ 干扰素 (IFN γ)的CD8阳性细胞的频数来反映CTL的杀伤活性。另设不加APC刺激的CD8阳性细胞 ,加IL 2培养作为对照组。结果本组 2 5例HCC患者中 9例HLA A2阳性表达 ;9例中 3例MAGE 3基因mRNA阳性表达 ,6例阴性表达。培养开始时 ,效应细胞为 1 0× 10 6/孔 ,2周后细胞增殖 4～ 6倍。分泌IFN γ的CD8阳性细胞的频数 ,3例MAGE 3阳性患者平均为 2 2 0 % ,6例阴性患者为 0 5 % ,两组间差异有非常显著意义 (P <0 0 1)。结论本实验结果表明 ,应用MAGE 3抗原肽 ,能在体外从HCC患者     

New epitope peptides derived from parathyroid hormone-related protein which have the capacity to induce prostate cancer-reactive cytotoxic T lymphocytes in HLA-A2+ prostate cancer patients

BACKGROUND: Parathyroid hormone-related protein (PTHrP) is produced by cancer cells and has been suggested to be responsible for malignancy-associated hypercalcemia and osteolysis after bone metatsases. Therefore, PTHrP is a promising target in the treatment of metastatic prostate cancer. METHODS: Seven PTHrP-derived peptides were prepared based on the HLA-A2 binding motif. These peptide candidates were screened by their ability to induce peptide-specific cytotoxic T lymphocytes (CTLs), and their ability to be recognized by immunoglobulin G (IgG). RESULTS: Both the PTHrP59-67 and PTHrP42-51 peptides were found to efficiently induce peptide-specific CTLs from peripheral blood mononuclear cells of HLA-A2+ prostate cancer patients with several HLA-A2 subtypes. These CTLs showed HLA-A2-restricted cytotoxicity toward prostate cancer cells. IgG reactive to the PTHrP42-51 peptide was frequently detected in prostate cancer patients. CONCLUSIONS: These results indicate that these two new PTHrP peptides will be useful in the peptide-based immunotherapy of HLA-A2+ prostate cancer patients, especially those with bone metastases.     

Vaccination of hormone-refractory prostate cancer patients with peptide cocktail-loaded dendritic cells: results of a phase I clinical trial

BACKGROUND: Immunotherapies might represent promising alternatives for the treatment of patients with hormone-refractory prostate cancer (HRPC). In a Phase I clinical trial, we evaluated a vaccination with dendritic cells (DCs) loaded with a cocktail consisting of HLA-A*0201-restricted peptides derived from five different prostate cancer-associated antigens [prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), survivin, prostein, transient receptor potential p8 (trp-p8)]. METHODS: Eight HRPC patients received a total of four vaccinations every other week. Clinical and immunological responses were monitored by the determination of the serum PSA levels and by enzyme linked immunospot (ELISPOT) analyses, respectively. RESULTS: Apart from local skin reactions no side effects were noted. One patient displayed a partial response (PR; PSA decrease >50%) and three other patients showed stable PSA values or decelerated PSA increases. In ELISPOT analyses, three of four PSA responders also showed antigen-specific CD8+ T-cell activation against prostein, survivin, and PSMA. CONCLUSIONS: The described protocol represents a safe and feasible concept for the induction of clinical and immunological responses. The application of a peptide cocktail-derived from different antigens as a novel treatment modality is supposed to allow for the genetic and biologic heterogeneity of PCa.