从卵巢癌cDNA文库中筛选新的肿瘤标志物

采用SEREX法筛选了自行构建的中国人卵巢癌cDNA表达文库 ,得到 2 7个阳性克隆 ,其中 3个为全长cDNA。序列分析和同源性比对结果表明所获得的 2 7个克隆分属于分化抗原、细胞结构蛋白及功能未知三类。选择其中一个全长cDNAMY OVA 13(该基因编码的蛋白是MAD2 ) ,利用基因工程方法克隆、表达和纯化得到目的蛋白 ,采用点杂交方法检测了MY OVA 13目的蛋白与正常人和肿瘤患者中的血清学反应。MY OVA 13抗体只在肿瘤组中检测到 (5 / 74 ) ,在正常组中均为阴性 ;间接ELISA测定结果显示 ,MY OVA 13抗体的水平在肿瘤组中均高于正常组 ,其中肝癌和前列腺癌组与正常组相比 ,在统计学上有显著差异 ,P值分别 <0 0 1和 <0 0 5。我们的初步结果表明MY OVA 13及其抗体具有一定的肿瘤特异性 ,有可能作为肿瘤诊断的标志物     

Identification of Antigen-Specific IgG in Sera from Patients with Chronic Prostatitis

Antigen-specific vaccines are one of several molecularly targeted approaches under investigation as possible treatments for prostate cancer. Important to the development of vaccines is the identification of appropriate target antigens. We hypothesized that antigens of the prostate might be identified in patients with the chronic prostatitis/pelvic pain syndrome, a syndrome for which an autoimmune pathology has been proposed. Such antigens might represent naturally recognized target antigens of the prostate that could be investigated in the future as prostate tumor antigens. In this report, we used SEREX to identify proteins expressed in a prostate cDNA expression library recognized by IgG from the sera of patients with chronic prostatitis. Candidate proteins were evaluated using a panel of sera from 62 subjects with symptomatic prostatitis and 71 control male blood donors. We identified one protein that was recognized primarily in sera from subjects with prostatitis compared with controls. MAD-PRO-34, a nucleolar autoantigen, was recognized in 6/62 subjects and 0/71 controls (p = 0.00897). This protein had previously been identified as an autoantigen in patients with prostate cancer. In addition, the NY-CO-7 protein was recognized in 9/62 subjects and 3/71 controls (p = 0.0654). Two subjects had IgG specific for both the MAD-PRO-34 and NY-CO-7 gene products. Our results demonstrate that some patients with the chronic prostatitis/pelvic pain syndrome have autoantibodies to specific proteins. Proteins identified, and MAD-PRO-34 in particular, could be further investigated as potential prostate tumor antigens.     

类风湿关节炎滑膜组织cDNA文库的构建及自身抗原的筛选和鉴定

目的 通过SEREX（serological analysis of recombinant cDNA expression library）技术对类风湿关节炎滑膜组织cDNA文库进行初步筛选,得到新的类风湿关节炎自身抗原.方法 利用SMART技术构建类风湿关节炎患者滑膜组织cDNA文库,并对该文库进行SEREX筛选,获得特异性结合阳性克隆,对阳性克隆同源性分析.然后进行酶联免疫吸附实验（ELISA）验证,分析该阳性克隆对于类风湿关节炎诊断临床敏感度和特异度.结果 构建cDNA文库滴度约为6.89×10^6 pfu/mL,筛选文库获得12个阳性克隆,同源性分析3个阳性克隆插入片段为已知蛋白,分别是：Filaggrin、SERPINE2和p68.进行血清学初步验证,抗-Filaggrin和抗-SERPINE2具有高敏感度和低特异度,而抗-p68具有高敏感度和高特异度.结论 抗-Filaggrin、抗-SERPINE2和抗-p68对于类风湿关节炎的诊断具有不同的敏感度和特异度.     

Toward a more complete recognition of immunoreactive antigens in squamous cell lung carcinoma

There is very limited knowledge about the antibody response against tumor-expressed antigens in lung cancer. To arrive at a more complete picture of lung cancer antigens, we generated 2 cDNA libraries from squamous cell lung carcinoma and isolated 15 immunogenic antigens using autologous sera. Among the antigens most frequently identified were the lymphoid blast crisis oncogene (LBC), an unknown hypothetical protein and the p53-binding protein (TP53 BP), which have already been associated with tumor development. Of the immunogenic antigens, 6 map to chromosomes that are frequently altered in squamous cell lung carcinoma. SEREX database analysis showed that 7 of the identified immunogenic antigens have been associated with the humoral immune response in other human tumors. Screening with heterologous sera of patients with lung carcinoma identified 4 antigens, including human protein kinase C and TP53 BP, which have also been found by autologous screening. Only 1 of the 15 identified antigens reacted with any of the 36 control sera, which were taken from individuals without known disease. Sera from adenocarcinoma and large cell carcinoma of the lung were not reactive for the antigens. In summary, using 2 newly established cDNA libraries, we isolated 15 novel antigens, which were subsequently evaluated for the frequency of their corresponding antibodies in autologous, normal and heterologous sera; their chromosomal localization; and their correlation with survival after surgery.     

Serologic analysis of ovarian tumor antigens reveals a bias toward antigens encoded on 17q

We utilized SEREX immunoscreening to identify a set of novel tumor antigens that are associated with human serous ovarian cancer and may prove useful for the early detection and treatment of this disease. Extensive screening with a panel of sera from 25 late-stage ovarian cancer patients against 3 independent cDNA libraries identified a set of 9 antigens that were immunogenic in more than 1 patient and not in a panel of 20-45 normal female serum donors. These antigens include p53, NY-ESO-1, UBQLN1, HOXB6, TOP2A, putative helicase-RUVBL (RUVBL), HMBA-inducible (HEXIM1), DDX5 and HDCMA. Ten of 25 ovarian cancer patients (40%) expressed serum IgG to at least 1 of these antigens, while 14% (4/25) had antibodies to 2 or more antigens. Unexpectedly, 4 antigens identified in this screen, DDX5, HEXIM1, TOP2A and HOXB6, are encoded within a region of 17q that also includes the genes for HER2/neu, Homeobox-B7 and BRCA1. Real-time RT-PCR analysis showed that mRNA for HER2/neu and 3 SEREX-defined antigens, TOP2A, HOXB6 and DDX5, was more abundant in ovarian tumors than most normal tissues, including normal and benign ovarian tissues, suggesting that elevated expression of genes encoded within this region of chromosome 17 is a common event in ovarian tumors. Thus, these abnormal expression patterns combined with the endogenous immune response suggests that these antigens represent potential targets for immunotherapy.     

Molecular characterization of virus-induced autoantibody responses

Here we present a comprehensive molecular mapping of virus-induced autoimmune B cell responses obtained by serological identification of antigens by recombinant expression cloning analysis. Immunoscreening of cDNA expression libraries of various organs (lung, liver, and spleen) using sera from mice infected with cytopathic (vaccinia virus [VV]) or noncytopathic (lymphocytic choriomeningitis virus [LCMV]) viruses revealed a broad specificity of the elicited autoantibody response. Interestingly, the majority of the identified autoantigens have been previously described as autoantigens in humans. We found that induction of virus-induced autoantibodies of the immunoglobulin G class largely depends on the CD40-CD40L-mediated interaction between T and B cells. Furthermore, antibody titers against a number of autoantigens were comparable to the concomitantly induced antiviral antibody response. Comparison of serum reactivity against a selected panel of autoantigens after infection with VV, LCMV, or vesicular stomatitis virus showed that the different virus infections triggered distinct autoantibody responses, suggesting that virus infections may leave specific "autoantibody fingerprints" in the infected host.     

Seroreactivity against MAGE-A and LAGE-1 proteins in melanoma patients

BACKGROUND: Cancer-testis antigens exemplify a growing number of tumour antigens which are expressed in a variety of malignancies, but not in normal tissues other than germ cells, primarily those of the testis. OBJECTIVES: To investigate the humoral response to known cancer-testis antigens in melanoma patients. METHODS: We used phage clones coding for seven different melanoma antigens MAGE-A or LAGE-1A proteins. These clones were isolated using the newly developed DNA hybridization analysis of recombinantly expressed cDNA libraries (HYREX) approach. HYREX combines the advantage of a nonradioactive library screening method with the possibility of subsequently analysing the serological response to the recombinant proteins. We isolated clones coding for MAGE-A1, -A3, -A4b, -A6, -A9 and -A12, as well as LAGE-1A. Additionally, we correlated gene expression and seroreactivity. RESULTS: Between 13% and 27% of sera (n = 15) were reactive against individual tumour antigens. We found the presence of specific antibodies was, with only two exceptions, generally correlated with mRNA expression of the antigen within cell lines derived from the same patient. While cross-reactivity of patients' IgG might play a role in these cases, antibodies from patients' sera were able to distinguish even the closely related MAGE-A3 and -A6. In general, the mRNA expression frequency was higher than the detected IgG responses. CONCLUSIONS: Antibody recognition of specific tumour antigens by patients' sera may be used for evaluating the possible immunogenicity of new antigens; serological tests could be used for tumour monitoring purposes.     

Novel tumor antigens elicit anti-tumor humoral immune reactions in a subset of patients with polycythemia vera

We attempted to determine whether the immune reactions elicited by aberrantly expressed testis antigens contribute to the beneficial responses to interferon (IFN)-alpha therapy and other therapies in patients with polycythemia vera (PV). We screened a human testis cDNA library using SEREX (serological analysis of tumor antigens by screening an expression cDNA library with sera from three patients with PV who had undergone IFN-alpha-induced or other therapeutics-induced remission). We identified two novel PV associated tumor antigens, PV65 (eIF-2alpha) and PV13 (protamine 2). These 2 antigens elicited IgG antibody reactions in a subset of PV patients but not in healthy donors, suggesting that they are authentic tumor antigens. Increased phosphorylation of PV65 in response to stimulation of IFN-alpha, and upregulation of PV13 in tumor cells might enhance their abilities in elicitation of immune reactions in patients. These findings provide new insights into the mechanism underlying the regulation of the self-antigen repertoire in eliciting anti-tumor immune reactions in patients with polycythemia vera, and suggest their potential as the targets of novel immunotherapy.     

Cancer Testis Antigen,NOL4, Is an Immunogenic Antigen Specifically Expressed in Small-Cell Lung Cancer

To identify cancer/testis (CT) antigens and immunogenic proteins, immunoscreening of testicular and small-cell lung cancer cell line NCI-H889 cDNA libraries was performed using serum obtained from a small-cell lung cancer (SCLC) patient. We obtained 113 positive cDNA clones comprised of 74 different genes, designated KP-SCLC-1 through KP-SCLC-74. Of these genes, 59 genes were found to be related to cancers by EMBASE analysis. Three of these antigens, including KP-SCLC-29 (NOL4), KP-SCLC-59 (CCDC83), and KP-SCLC-69 (KIF20B), were CT antigens. RT-PCR and western blot analysis showed that NOL4 was frequently present in small-cell lung cancer cell lines (8/9, 8/9). In addition, NOL4 mRNA was weakly, or at a low frequency, or not detected in various cancer cell lines. Our results reveal that NOL4 was expressed at protein levels in small-cell lung cancer tissues (10/10) but not detected in lung adenocarcinoma and squamous cell carcinoma by immunohistochemical analysis. Serological response to NOL4 was also evaluated by western blot assay using NOL4 recombinant protein. A humoral response against NOL4 proteins was detected in 75% (33/44) of small-cell lung cancer patients and in 65% (13/20) of healthy donors by a serological western blot assay. These data suggest that NOL4 is a specific target that may be useful for diagnosis and immunotherapy in SCLC.     

Anti‐FIRΔexon2 autoantibody as a novel indicator for better overall survival in gastric cancer

There is no clinically available biomarker for efficiently indicating the overall survival or therapy response of gastric cancer (GC). The autoantibodies (Abs) in the sera of anti‐far‐upstream element‐binding protein‐interacting repressor‐lacking exon2 (FIRΔexon2), anti‐sorting nexin 15, and anti‐spermatogenesis and oogenesis–specific basic helix–loop–helix 1 were markedly higher in GC patients than in healthy donors (HDs). These Abs were identified by large‐scale serological identification of antigens by recombinant cDNA expression cloning screenings and their expression levels were evaluated by amplified luminescence proximity homogeneous assay. In particular, compared with age‐matched HDs, the level of anti‐FIRΔexon2 Abs in GC patients was significantly higher (P < .001). The Spearman''s rank correlation analysis between anti‐FIRΔexon2 Abs and clinically available tumor markers such as carcinoembryonic antigen (CEA) was statistically insignificant, indicating that FIRΔexon2 Abs is an independent biomarker. We performed receiver‐operating curve analysis to evaluate the anti‐FIRΔexon2 Ab as a candidate biomarker with CEA and carbohydrate antigen 19‐9 (CA19‐9). The overall survival of GC patients with high anti‐FIRΔexon2 Abs titer was significantly favorable (P = .04) than that of GC patients who were below detection level of anti‐FIRΔexon2 Abs. However, clinical stages were not apparently correlated with the levels of anti‐FIRΔexon2 Ab, CEA, and CA19‐9. In conclusion, anti‐FIRΔexon2 Abs detected in GC patients is a potential biomarker for monitoring a better prognosis. Hence, anti‐FIRΔexon2 Abs is a promising biomarker for indicating better overall survival of gastric cancer patients.