肝癌相关肿瘤抗原AFP新的HLA-A2/A24 限制性CTL表位的预测及分析

目的:预测肝癌肿瘤抗原AFP新的HLA-A2/A24限制性CTL表位,为肝癌CTL表位肽的筛选及鉴定提供依据.方法:选择与肝癌密切相关的肿瘤抗原AFP为研究目标,采用NetCTL 1.2 Server远程预测系统进行HLA-A2/A24限制性CTL表位预测.结果:共预测出与肝癌相关的肿瘤相关抗原AFP的8个HLA-A2 限制性CTL表位,13个HLA-A24限制性CTL 表位.结论:应用NetCTL 1.2 Server法预测CTL表位是一种高效准确的预测方法.所预测的肿瘤抗原AFP的HLA-A2/24 限制性CTL表位经实验筛选、鉴定后,可为肝癌治疗性疫苗的制备选择合适的CTL表位提供依据.     

肿瘤相关抗原CT45的HLA-A2/A3限制性CTL表位的预测

目的:预测肿瘤相关抗原CT45的HLA-A2/A3限制性CTL表位,为CTL表位肽的合成和活性筛选提供依据。方法:基于新近发现的CT45抗原的氨基酸序列,用NetCTL 1.2 Server人工神经网络法远程预测系统进行HLA-A2/A3限制性CTL表位预测打分,挑选出分值较高的作为候选表位。结果:共预测出了5个潜在的HLA-A2限制性CTL表位九肽,15个潜在的HLA-A3限制性CTL表位九肽,所有这些表位均为首次报道。结论:NetCTL 1.2 Server人工神经网络法能高效的预测CTL表位肽,基于新近发现的CT45抗原,我们通过该系统成功预测出了5个潜在的HLA-A2限制性CTL表位,15个潜在的HLA-A3限制性CTL表位,可以对其进行进一步的研究。     

苦瓜蛋白MAP30的HLA—A2／A3限制性CTL表位预测

目的：预测苦瓜蛋白MAP30的HLA—A2／A3限制性CTL表位。方法：应用lasergene7．0和NetCTL-1．2Server对MAP30进行HLA—A2／A3限制性CTL表位预测。结果：MAP30的二级结构含有8个d-螺旋和9个B-折叠股;柔韧性良好氨基酸区域为19—40、99—146、166—273;亲水性指数较高区域为22—45、97—114、117～148、180—198、224—257、265—275;抗原性指数较高区域为19—28、63—71、98-114、118—123、224—246、248—260、262—274;有30个可能CTL表位,综合分析后获得2个HIJA—A2和5个HLA—A3限制性CTL表位。结论：MAP30具有潜在的7个HLA—A2／A3限制性CTL表位。     

肝癌MAGE-n抗原HLA-A2限制性细胞毒性T细胞表位的预测

目的 预测肝癌MAGE—n抗原HLA—A2限制性细胞毒性T淋巴细胞(CTL)表位。方法 应用SYFPEITHI超基序多项式法远程预测系统和量化基序多项式法结合的预测方法，筛选MAGE—n抗原HLA—A2限制性CTL表位。结果 筛选到MAGE—n抗原5个HLA—A2限制性CTL表位(九肽)。结论 SYFPEITHI超基序法和量化基序多项式法结合的预测方法是一种高效和准确的表位预测方法。     

肿瘤抗原MAGE-12新的HLA-A2限制性细胞毒性T细胞表位的预测

目的预测黑色素瘤抗原MAGE-12的HLA—A2限制性CTL表位。方法采用SYFPEITHI超基序远程预测系统与量化基序多项式法、延展基序联合应用，筛选MAGE-12抗原HLA—A0201限制性CTL表位。结果共预测出5个MAGE-12抗原HLA—A2限制性CTL表位。结论超基序法与量化基序，延展基序联合应用可以提高预测效率，为实验方法探索MAGE-12的表位提供有用线索。     

癌-睾丸抗原SSX-3的HLA-A2/A3限制性CTL广谱表位的预测

目的:预测基于癌-睾丸抗原SSX-3的HLA-A2/A3限制性CTL广谱表位.方法: 采用MULTIPRED在线预测系统分别针对HLA-A2超型和HLA-A3超型对SSX-3序列进行CTL表位预测,其中对两种超型分值均较高的表位被初步认定为广谱表位;然后,运用NetCTL 1.2在线预测系统对初步认定的广谱表位进行蛋白酶体裂解和TAP转运效率预测进一步筛选.结果: 共预测出9个SSX-3抗原的HLA-A2/A3限制性广谱表位.结论: MULTIPRED与NetCTL 1.2在线预测系统相结合可以高效的预测广谱CTL表位;本报道为SSX-3的广谱CTL表位的实验研究提供了重要的理论依据.     

原癌基因Bmi-1 B细胞抗原表位及 HLA I类限制性CTL表位的生物信息学分析

目的:预测原癌基因Bmi-1(B-cell-specific Moloney murine leukemia virus insertion site 1)的B细胞抗原表位及HLA I类限制性细胞毒性T细胞（cytotoxic T cell）表位。方法:采用Ellipro(http://tools.immuneepitope.org/ellipro/)预测Bmi-1蛋白中可能存在的线性B细胞表位和构象B细胞表位；采用ProtParam、NetCTL等软件和方法预测Bmi-1中可能存在的HLA I类限制性CTL表位。结果:Bmi-1蛋白中含有6个线性B细胞表位和8个构象B细胞表位；结合HLA结合力、蛋白酶体切割效率和TAP转运效率，经过NetCTL程序预测表明:针对不同的HLA I类分子，原癌基因Bmi-1中都存在多个HLA I类分子的限制性CTL表位。结论:综合运用多个程序预测了原癌基因Bmi-1中的B细胞抗原表位及HLA I类分子的限制性CTL表位，为下一步开展针对Bmi-1的免疫靶向治疗等研究打下了基础。     

苦瓜蛋白MAP30的HLA-A2/A3限制性CTL表位预测

目的:预测苦瓜蛋白MAP30的HLA - A2/A3限制性CTL表位.方法:应用lasergene 7.0和NetCTL-1.2 Server对MAP30进行HLA - A2 /A3限制性CTL表位预测.结果:MAP30的二级结构含有8个α-螺旋和9个β-折叠股;柔韧性良好氨基酸区域为19-40、99-146、166-273;亲水性指数较高区域为22-45、97-114、117-148、180-198、224-257、265-275;抗原性指数较高区域为19-28、63-71、98-114、118-123、224-246、248-260、262-274;有30个可能CTL表位,综合分析后获得2个HLA - A2和5个HLA - A3限制性CTL表位.结论:MAP30具有潜在的7个HLA-A2/A3限制性CTL表位.     

OVA66抗原肽与HLA-A2分子结合性和结合稳定性的研究

目的：建立利用T2细胞体外筛选肿瘤相关抗原OVA66中HLAA2限制性抗原肽表位的实验方法。方法：应用计算机预测获得OVA66抗原中多个不同分值的九肽分子，在不同浓度(0.2 μg/ml,2 μg/ml,5 μg/ml和20 μg/ml)下与T2细胞共同孵育4 h，并分别在其后的0，2,4 h测定T2细胞表面HLAA2分子的平均表达强度，抗原肽与HLAA2的亲和力以20％MFImax时的抗原肽作用浓度表示；抗原肽与HLAA2分子结合稳定性以MFI(4 h)/MFI(0 h)的百分比表示。结果：经计算机预测所获得的分值不同的4种候选OVA66抗原肽表位与T2细胞表面HLAA*0201的结合能力和结合稳定性显示出差别，并且与预测的分值不尽符合，其中L235和L238与HLAA *0201的结合力优于L236和L237。 结论：利用T2细胞可以在体外初步筛选获得与HLAA *0201具有高亲和力的抗原肽。     

乳腺癌相关基因环氧化酶2（COX-2）编码蛋白的主要特性与抗原表位生物信息学分析

目的：通过生物信息学分析人乳腺癌相关基因环氧化酶2（Cycloxygenase2,COX-2）编码蛋白的主要特性与抗原表位,筛选该蛋白的T／B细胞联合表位。方法：利用蛋白分析专家系统（ExPASy）提供的Prot—Param、SignalP、SOSUI、TMHMM、MotifScan,NCBI上的ORFfinder、Bcepred、SYFPElTHI、NetCTL等生物信息学在线分析程序,结合GeneRunner、DNAMAN等生物信息学软件,分析、预测COX-2蛋白的理化性质、信号肽、可溶性、表面可及性、可塑性,跨膜区,翻译后修饰位点、亲（疏）水性、二级结构、T／B细胞抗原表位等。结果：该蛋白由604个氨基酸组成,分子式为C3126H4787N8230883S28,分子质量单位为68．924kDa,等电点理论值为7．27,波长280nm时的吸光度（A）值为1．073,半衰期为30h,有7个表面可及性参数≥1．9的区域、4个亲水性参数得分≥1．9的区域、3个柔韧性参数得分≥2的区域、26个翻译后修饰位点、23个潜在B细胞表位、3个CTL表位和辅助性T细胞联合表位、2个T／B细胞联合表位,为可溶性表达蛋白。结论：人乳腺癌相关抗原COX-2基因编码的COX-2蛋白为可溶性表达蛋白,2个T／B细胞联合表位,可作为乳腺癌免疫学治疗的重要靶点。     

Prediction and analysis of HLA-A2/A24-restricted cytotoxic T-lymphocyte epitopes of the tumor antigen MAGE-n using the artificial neural networks method on NetCTL1.2 Server

Cancer immunotherapy has become one of the most important therapeutic approaches to cancer in the past two decades. Tumor antigen-derived peptides have been widely used to elicit tumor-specific cytotoxic T lymphocytes (CTLs). Antigen-specific CTLs induced by MAGE-derived peptides have proven to be highly efficacious in the prevention and treatment of various types of tumor. MAGE-n is a new member of the MAGE gene family and has been shown to be closely associated with hepatocellular carcinoma. It is highly homologous to the MAGE-A gene subfamily, particularly to MAGE-3 (93%). MAGE-n-derived peptide QLVFGIEVV is a novel HLA-A2.1-restricted CTL epitope that induces MAGE-n-specific CTLs in vitro. Identification of these CTL epitopes may lead to clinical applications of these peptides as cancer vaccines for patients with MAGE-n(+)/HLA-A2(+) tumors. In the present study, HLA-A/A24-restricted CTL epitopes of antigen MAGE-n were predicted using the NetCTL1.2 Server on the web, COMB >0.85. The results showed that the NetCTL1.2 Server prediction method improved prediction efficacy and accuracy. Additionally, 8 HLA-A2- and 9 HLA-A24-restricted CTL epitope candidates (nonamers) derived from the tumor antigen MAGE-n were predicted. These nonamers, following identification via experimentation, may contribute to the development of potential antigen peptide tumor vaccines.     

Identification of alpha-fetoprotein-derived peptides recognized by cytotoxic T lymphocytes in HLA-A24+ patients with hepatocellular carcinoma

Alpha-fetoprotein (AFP) has been proposed as a potential target forT-cell-based immunotherapy for hepatocellular carcinoma (HCC), but the number of its epitopes that have been identified is limited and the status of AFP-specific immunological responses in HCC patients has not been well-characterized. To address the issue, we examined the possibility of inducing AFP-specific cytotoxic T cells (CTLs) using novel HLA-A*2402-restricted T-cell epitopes (HLA, human leukocyte antigen) derived from AFP and then analyzed the relationship between its frequency of occurrence and clinical features associated with patients having HCC. Five AFP-derived peptides containing HLA-A*2402 binding motifs and showing high binding affinity to HLA-A*2402 induced CTLs to produce IFN-gamma and kill an AFP-producing hepatoma cell line. The frequency of AFP-specific CTLs was 30-190 per 1 x 10(6) peripheral blood mononuclear cells, which was the same as that of other immunogenic cancer associated antigen-derived epitopes. Analyses of the relationships between AFP-specific CTL responses and clinical features of patients with HCC revealed that AFP epitopes were more frequently recognized by CTLs in patients with advanced HCC correlating to tumor factors or the stage of TNM classification. The analyses of CTL responses before and after HCC treatments showed that the treatments changed the frequency of AFP-specific CTLs. In conclusion, we identified five HLA-A*2402-restricted T-cell epitopes derived from AFP. The newly identified AFP epitopes could be a valuable component of HCC immunotherapy and for analyzing host immune responses to HCC.     

Identification of HLA-A2-restricted CTL epitope encoded by the MAGE-n gene of human hepatocellular carcinoma

BACKGROUND: Identification of the cytotoxic T lymphocytes (CTL) restricted epitopes of tumor antigens opens up possibilities of developing a new cancer vaccine. For the MAGE-n has been demonstrated closely associated with hepatocellular carcinoma (HCC) and HLA-A2.1 is found in over 50% of HCC patients in China, we aim at identifying MAGE-n-encoded peptide presented by HLA-A2.1. MATERIALS: A HLA-A2.1-restricted CTL epitope was identified by using an improved "reverse immunology" strategy: (a) computer-based epitope prediction from the amino acid sequence of MAGE-n antigen; (b) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A2.1 molecule; (c) stimulation of primary T-cell response against the predicted peptides in vitro; and (d) testing of the induced CTLs toward HCC cells expressing MAGE-n antigen and HLA-A2.1. RESULTS: Of the five tested peptides, effectors induced by a peptide of MAGE-n at residue position 159-167(QLVFGIEVV) lysed HCC cells expressing both MAGE-n and HLA-A2.1. Our results indicated that peptide QLVFGIEVV was a new HLA-A2.1-restricted CTL epitope capable of inducing MAGE-n specific CTLs in vitro. CONCLUSIONS: Identification of the MAGE-n /HLA-A2.1 peptide QLVFGIEVV may facilitate peptide-based specific immunotherapy for HCC. The combination of epitope prediction, epitope reconstruction method and immunological methods can improve the efficiency and accuracy of CTL epitope studies.     

肝癌中HLA抗原表达及临床意义

目的探讨HLA 抗原在肝癌中的表达水平及与病程、预后等的关系.方法应用5种HLA 及相关分子单抗,采用免疫组织化学方法(ABC法)对93例肝细胞肝癌及癌旁组织的石蜡切片进行检测.结果 HLA 类抗原在癌旁正常肝细胞中不表达或低表达,在癌细胞中表达明显增强;肝癌中HLA 类抗原表达增强的患者生存率较其他患者高,肝癌中I类抗原表达阳性的患者较表达阴性的患者生存率高.未发现肝癌细胞HLA 类抗原表达的增强与年龄、AFP、HBsAg、肝硬化、瘤体大小及病理分级有关.HLA I类抗原在肝癌及癌旁细胞中均表达较弱或不表达.结论 HLA 类抗原在肝癌中表达较癌旁细胞增强,并有利于患者的预后.肝癌中HLA 类抗原表达增强这种有别于其他肿瘤的分子机制及意义等问题还需进一步研究.     

Dendritic cells pulsed with alpha-fetoprotein and mutant P53 fused gene induce bi-targeted cytotoxic T lymphocyte response against hepatic carcinoma

Dendritic cell (DC)-based immunotherapy is rapidly emerging as a promising treatment in cancer therapy. We had previously shown that DC pulsed with either defined mRNA of tumor antigen (Ag) such as α-fetoprotein (AFP), or total RNA of hepatocellular carcinoma (HCC) could elicit Ag-specific cytotoxic T lymphocyte (CTL) response. Therefore, we suggested a novel DC-based therapeutic method, in which DCs derived from CD34⁺ cells enriched peripheral blood mononuclear cells were pulsed with liposome-coated AFP and mutant P53 (mtP53) fused gene pEGFP-C3/AFP-mtP53 to induce bi-targeted specific CTL responses against HCC. Three different genotype HCC cell lines, HepG2 (human histocompatibility leukocyte antigens (HLA) A2 positive, AFP expressing positive, P53 expressing negative), SMMC7721 (HLA A2 positive, neither AFP nor P53 expressing positive), and HMCC97 (HLA A2 positive, both AFP and P53 expressing positive) were selected as targets for CTL responses. An important finding was that DCs pulsed with the liposome-coated fused gene could evoke more intensive bi-targeted Ag-specific CTL responses against HMCC97 than DCs pulsed with either AFP or P53 single gene ( P <  0.05). This experimental therapeutic model provides a new promising cytotherapeutic approach, in that DCs pulsed with the fused gene of different Ags might induce more extensive multitargeted antitumor immunity. ( Cancer Sci 2008; 99: 1420–1426)     

Identification of HLA-A24-restricted CTL epitope from cancer-testis antigen, NY-ESO-1, and induction of a specific antitumor immune response.

PURPOSE: For the development of peptide-based, cancer-specific immunotherapy, the identification of CTL epitopes from additional tumor antigens is very important. NY-ESO-1, a cancer-testis antigen, is considered to be a promising target of tumor-specific immunotherapy. Because HLA-A24-expressing individuals cover >60% in the population of Japan, we aim at identifying NY-ESO-1-encoded peptide presented by HLA-A24. EXPERIMENTAL DESIGN: In our study, a HLA-A24-restricted CTL epitope was identified by using the following four-step procedure: (a) computer-based epitope prediction from the amino acid sequence of NY-ESO-1 antigen; (b) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A24 molecule; (c) stimulation of primary T-cell response against the predicted peptides in vitro; and (d) testing of the induced CTLs toward various carcinoma cells expressing NY-ESO-1 antigen and HLA-A24. RESULTS: Of the tested peptides, effectors induced by a peptide of NY-ESO-1 at residue position 158-166 lysed three kinds of carcinoma cells expressing both NY-ESO-1 and HLA-A24. Our results indicate that peptide NY-ESO-1 (158-166) (LLMWITQCF) is a new HLA-A24-restricted CTL epitope capable of inducing NY-ESO-1-specific CTLs in vitro mediating HLA class I-restricted manner. CONCLUSIONS: We identified a novel HLA-A24-restricted NY-ESO-1-derived epitope peptide (LLMWITQCF) that could induce specific CTLs from the peripheral blood mononuclear cells of HLA-A24(+) healthy donors. This peptide would be useful in further evaluating the clinical utility of peptide-based, cancer-specific immunotherapy against various histological tumors.