Identification of Tumor-Associated Antigens from Medullary Breast Carcinoma by a Modified SEREX Approach

Medullary breast carcinoma (MBC) is a relatively rare malignancy with heavy lymphocytic infiltration that despite cytologically anaplastic features and high mitotic index has more favorable prognosis than other types of breast cancer. Lymphocytic infiltration of tumors reflects ongoing immune response against tumor antigens which could represent a great interest as potential targets for cancer immunotherapy. The search for MBC antigens by SEREX methodology has not been successful due to a very high titer of false positive clones, representing immunoglobulin genes. Here, we describe a novel approach for generating cDNA expression libraries from MBC tumor samples which are depleted of IgG cDNA clones and, therefore, are suitable for the identification of novel tumor-associated antigens (TAA) by SEREX approach. Modified methodology allowed us to isolate a panel of known and novel TAA which are currently under further investigation.     

Serological study of a repertoire of human cancer antigens and autoantigens

The spectrum of human antigens allows a monitoring of various pathological processes such as autoimmune disorders and tumorigenesis. Serological analysis of cDNA expression libraries (SEREX) is now used to search for new cancer-associated antigens, which are potential diagnostic markers or targets for immunotherapy of cancer. The results obtained for several solid tumors are reviewed. Groups of antigens detectable by SEREX, causes of immunogenicity of autoantigens, and prospective implication of antigens in diagnostics and monitoring of cancer are discussed.     

Serological Analysis of the Repertoire of Human Cancer Antigens and Autoantigens

The spectrum of human antigens allows a monitoring of various pathological processes such as autoimmune disorders and tumorigenesis. Serological analysis of cDNA expression libraries (SEREX) is now used to search for new cancer-associated antigens, which are potential diagnostic markers or targets for immunotherapy of cancer. The results obtained for several solid tumors are reviewed. Groups of antigens detectable by SEREX, causes of immunogenicity of autoantigens, and prospective implication of antigens in diagnostics and monitoring of cancer are discussed.     

多发性骨髓瘤细胞中一个新的Alu超基因家族成员的克隆与分析

根据Genebank收录的多发性骨髓瘤细胞株（ARH－77）表达上调EST AF497797设计引物,采用半定量RT－PCR证实了多发性骨髓瘤患者及正常人骨髓细胞中该expressed sequence tages(EST)存在表达差异。用EST AF497797作探针筛选人胚肾cDNA文库,获得cDNA克隆经测序并用生物信息学方法对该序列进行了初步分析。AF497797在多发性骨髓瘤患者骨髓中确有较高的表达,而在正常人骨髓细胞中低表达。获得的全长cDNA克隆序列长1248bp(Genebank登录号：AY094612）。生物信息学分析显示该片段全长cDNA编码44个氨基酸的蛋白产物且可能属于Alu家族成员。基因AY094612为一个在多发性骨髓瘤中表达上调的新基因,其改变可能与多发性骨髓瘤的发生与发展有关。     

Optimized serological isolation of lung-cancer-associated antigens from a yeast surface-expressed cDNA library

The technique of serological analysis of antigens by recombinant cDNA expression library (SEREX) uses autologous patient sera as a screening probe to isolate tumor-associated antigens for various tumor types. Isolation of tumor-associated antigens that are specifically reactive with patient sera, but not with normal sera, is important to avoid false-positive and autoimmunogenic antigens for the cancer immunotherapy. Here, we describe a selection methodology to isolate patient sera-specific antigens from a yeast surface-expressed cDNA library constructed from 15 patient lung tissues with non-small cell lung cancer (NSCLC). Several rounds of positive selection using patient sera alone as a screening probe isolated clones exhibiting comparable reactivity with both patient and normal sera. However, the combination of negative selection with allogeneic normal sera to remove antigens reactive with normal sera and subsequent positive selection with patient sera efficiently enriched patient sera-specific antigens. Using the selection methodology described here, we isolated 3 known and 5 unknown proteins, which have not been isolated previously, but and potentially associated with NSCLC.     

Identification of tumor antigens as potential target antigens for immunotherapy by serological expression cloning

The presence of tumor infiltrating T cells has been shown to be associated with a favorable prognosis in different tumor types. Several strategies have been developed to identify relevant tumor antigens which can be used for active immunotherapy strategies. The SEREX technique (serological analysis of cDNA expression libraries) identifies tumor antigens based on a spontaneous humoral immune response in cancer patients. This technique is not limited to tumor types that can be grown in cell culture or depends on established T cell clones recognizing the autologous tumor. Several steps of analysis are mandatory to evaluate SEREX-defined antigens before they become new target antigens for active immunotherapy: expression analysis; serological analysis with sera from tumor patients and normal individuals; identification of potential peptide epitopes for CD8 T cells and evaluation in T cell assays. This article summarizes our approach of antigen identification and evaluation giving the example of the recently cloned breast cancer antigen NY-BR-1.This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

Analysis of the antibody repertoire of lymphoma patients

Cancer testis or cancer germline antigens (CGA) are promising vaccine candidates because they are expressed only in malignant but not in normal tissues, except for germ cells in the testis. Since non-Hodgkin's lymphomas (NHL) express the known CGA at low frequencies, we aimed at increasing the number of CGA with frequent expression in NHL by screening a cDNA expression library derived from normal testis for reactivity with high-titered IgG antibodies in the sera of lymphoma patients using SEREX, the serological identification of antigens by recombinant cDNA expression cloning. The analysis of 1.6x10(6) clones with the sera of 25 lymphoma patients revealed 42 clones which coded for 23 antigens, 12 of which had already been included in the SEREX databank. Four cDNA clones coded for unknown and 19 for known genes. Three antigens reacted only with the serum by which they had been detected, 9 antigens reacted with the sera of several NHL patients, but not with that of healthy controls, and 11 antigens reacted with both normal and NHL sera. Most of the antigens were ubiquitously expressed. Only HOM-NHL-6, HOM-NHL-8, HOM-NHL-21 and HOM-NHL-23 showed a restricted expression pattern. HOM-NHL-6 and HOM-NHL-8 were homologous to the previously described CGA NY-ESO-1 and HOM-TES-14/SCP-1, respectively. HOM-NHL-21 was expressed in rare cases of lymphomas, but not in normal tissues except for testis and brain, while HOM-NHL-23 appeared to be a testis-specific antigen. In summary, using the antibody repertoire of these 25 NHL patients, no new CGA were detected. The number of CGA detectable by the classical SEREX approach appears to be limited, and novel strategies are necessary to identify antigens that can serve as a vaccine target in a broad spectrum of NHL patients.     

鼻咽癌组织cDNA文库的构建及抗原基因的筛选

构建人鼻咽癌组织cDNA文库,以SEREX方法从cDNA文库中筛选鼻咽癌抗原基因。采用确诊鼻咽癌患者新鲜活检癌组织构建cDNA文库,测定原始文库滴度,进行蓝白筛选以确定文库的重组率。以建库组织来源患者的自身血清,采用“一对一” 的血清学方法筛选所构建的cDNA文库,阳性克隆经PCR检测鉴定后进行序列分析。经测定原始文库滴度为7.28×10⁶pfu/mL,含3.64×10⁶个重组子,重组率为94%,扩增文库滴度为3.8×10⁹pfu/mL,cDNA插入片段大小在0.5～3.0kb之间。文库经三轮血清学筛选共获得23个阳性克隆,分别代表了16个独立的cDNA插入片段（抗原基因）。其中10个与已知基因高度同源,另外6个基因与GenBank中已知基因的部分同源,其中有3个是新基因。利用SMART技术构建了高质量的人鼻咽癌组织cDNA表达文库,有利于以cDNA文库为基础的进一步的实验研究。应用SEREX技术初步筛选鼻咽癌组织cDNA文库,共得到16个鼻咽癌相关抗原基因,其中有3个是新基因,可能为鼻咽癌的免疫学研究提供新的研究分子。     

The reverse proteomics for identification of tumor antigens

The identification of tumor antigens is essential for the development of anticancer therapeutic vaccines and clinical diagnosis of cancer. SEREX (serological analysis of recombinant cDNA expression libraries) has been used to identify such tumor antigens by screening sera of patients with cDNA expression libraries. SEREX-defined antigens provide markers for the diagnosis of cancers. Potential diagnostic values of these SEREX-defined antigens have been evaluated. SEREX is also a powerful method for the development of anticancer therapeutics. The development of anticancer vaccines requires that tumor antigens can elicit antigen-specific antibodies or T lymphocytes. More than 2000 antigens have been discovered by SEREX. Peptides derived from some of these antigens have been evaluated in clinical trials. This review provides information on the application of SEREX for identification of tumor-associated antigens (TAA) for the development of cancer diagnostics and anticancer therapeutics.     

Identification of BCP-20 (FBXO39) as a cancer/testis antigen from colon cancer patients by SEREX

Cancer/Testis (CT) antigens are considered promising target molecules for immunotherapy. To identify potential CT antigens, we performed immunoscreening of a testis cDNA library with sera from colon cancer patients by SEREX. We isolated 114 positive cDNA clones comprising 90 different antigens, designated BCP-1 through BCP-90. Quantitative real-time and conventional RT-PCR analysis showed that BCP-20, -33, and -41 antigens were expressed strongly only in a normal testis and detected in 22 cases (39%), 12 cases (21%), and 17 cases (30%), respectively, from 57 colon tumors. BCP-20 was also detected in various cancer cell lines including breast, colon, hepatoma, renal, thyroid anaplastic, ovary, sarcoma, and lung. By ELISA analysis, anti-BCP-20 antibody was detected in 3 of 50 colon cancer and 1 of 24 gastric cancer patients while healthy donors were three positive (3/50). But the BCP-20 antibody levels of patients with colon cancer showed significantly higher titers than those of healthy donors. These data suggest that the BCP-20 gene is a new CT antigen and may be useful for diagnosis and immunotherapy.     

Serological cloning of PARIS-1: a new TBC domain-containing, immunogenic tumor antigen from a prostate cancer cell line.

Identifying immunogenic tumor antigens plays a critical role in developing efficient diagnostic and therapeutic strategies for treatment of cancer. Using a recently developed technology, serological identification of antigens by recombinant expression cloning (SEREX), we identified a total of 8 genes whose expression elicited antibody responses in prostate cancer patients. Of the 8 genes, 5 represented known genes in the GenBank database, 2 were previously uncharacterized genes, and 1 showed sequence homology to a mouse gene. The sequence feature and the expression of one of the novel genes, prostate antigen recognized and identified by SEREX (PARIS-1), are determined in this study. The PARIS-1 cDNA is 3257 bp in length and contains a complete open reading frame of 2751 bp encoding for a primary translation product of 917 amino acids. Using Northern blot hybridization assay, we detected a single species of approximately 3.3 kb PARIS-1 mRNA that is differentially expressed in prostate normal and cancer cells. Western blot analysis confirmed the expression of the PARIS-1 protein in these cells. Structure analysis revealed that PARIS-1 protein contains a TBC domain that is conserved in the family of cell cycle-regulatory and Rab GTPase-activating proteins (Rab-GAP). Thus, the PARIS-1 protein may play a role in regulation of cell differentiation and growth or represent a new member of the Rab-GAP family.     

多发性骨髓瘤细胞中一个表达上调基因的克隆与分析

根据GenBank收录的多发性骨髓瘤细胞株 (ARH 77)表达上调ESTAF4 2 5 30 0设计引物 ,运用RT PCR检测了 5例多发性骨髓瘤患者及 4例正常人骨髓细胞中该EST的表达水平 .Northern印迹杂交分析该EST在多种组织中的表达 .进一步利用该EST作探针 ,筛选ARH 77cDNA文库 ,获得全长cDNA克隆 ,对该序列进行了分析 .结果显示 ,该EST在多发性骨髓瘤患者骨髓细胞中亦有较高的表达 ,而在正常人骨髓细胞中低表达 .经测序证实 ,该cDNA全长为 4 5 2bp(GenBank收录号 :AF4 87338) .预测其编码一个 5 7个氨基酸的小分子量蛋白质 ,属于与DNA复制有关的解旋酶 引物酶基因家族的新成员 .该基因在多发性骨髓瘤细胞中表达上调 ,其表达水平的改变可能与多发性骨髓瘤的发生与发展有关     

Characterisation of tumour-associated antigens in colon cancer

In order to search for clinically relevant cancer-associated genes and to define further the spectrum of immunogenic proteins, we applied SEREX (serological identification of antigens by recombinant expression cloning) to analyse genes expressed in colon adenocarcinoma. Eight different serum-reactive cDNA clones were isolated by immunoscreening from a colon cancer-derived cDNA expression library. mRNA expression studies showed that 2 of them, RHAMM and AD034, have a differential tissue distribution, and that 3 genes, NAP1L1, RHAMM and AD034, are overexpressed in tumours in comparison with the adjacent non-cancerous tissues. 5' RLM-RACE analysis of AD034, a sequence with a tyrosine kinase motif, revealed a frameshifting insertion of 32 bp, most likely generated by use of cryptic splice site in tumour-derived cDNA. Analysis of full-length RHAMM cDNA sequence revealed the presence of two splice variants, which are known to have a different sub-cellular localisation; expression of these splice variants is altered in colon cancer tissues. Serological responses to three antigens (C21ORF2, EPRS and NAP1L1) were found mainly in cancer patients' sera.     

MAGE-C1/CT7 and MAGE-C2/CT10 are frequently expressed in multiple myeloma and can be explored in combined immunotherapy for this malignancy

The exact function of MAGE-C1/CT7 and MAGE-C2/CT10 is not yet understood in multiple myeloma (MM). However, the homologs MAGE-C1/CT7 and MAGE-C2/CT10 genes encode highly immunogeneic cancer/testis antigens (CTAs) and can be potential targets for T cell-based immunotherapy. MAGE-C1/CT7 and MAGE-C2/CT10 mRNA expression were investigated in MM patients, solitary plasmacytomas, monoclonal gammopathies of undetermined significance (MGUS) and bone marrow (BM) aspirates from healthy donors by RT-PCR. MAGE-C1/CT7 and MAGE-C1/CT10 were expressed in 67 and 59 % of the 46 MM analyzed patients. At least one of the genes was expressed in 76 % of MM cases. Solitary plasmacytoma also showed MAGE-C1/CT7 and MAGE-C2/CT10 expression. MAGE-C1/CT7 and MAGE-C2/CT10 were not expressed in normal BM samples, showing restricted expression of these CTA genes in MM, solitary plasmacytoma and MGUS. In the present study, we found high expression of the homologs MAGE-C1/CT7 and MAGE-C2/CT10 in monoclonal gammopathies and speculate whether these genes might represent a valuable therapeutic option for myeloma, in particular for combined immunotherapy.     

Humoral detection of leukaemia-associated antigens in presentation acute myeloid leukaemia

The serological analysis of recombinant cDNA expression libraries (SEREX) technique was used to immunoscreen a testes cDNA expression library with sera from newly diagnosed acute myeloid leukaemia (AML) patients. We used a testis cDNA library to aid our identification of cancer-testis (CT) antigens. We identified 44 antigens which we further immunoscreened with sera from AML, chronic myeloid leukaemia (CML), and normal donors. Eight antigens were solely recognised by patient sera including the recently described CT antigen, PASD1, and the cancer-related SSX2 interacting protein, SSX2IP. RT-PCR analysis indicated that we had identified three antigens which were expressed in patient bone marrow (BM) and peripheral blood (PB) but not in normal donor samples (PASD1, SSX2IP, and GRINL1A). Real-time PCR (RQ-PCR) confirmed the restricted expression of PASD1 in normal donor organs. Antigen presentation assays using monocyte-derived dendritic cells (mo-DCs) showed that PASD1 could stimulate autologous T-cell responses, suggesting that PASD1 could be a promising target for future immunotherapy clinical trials.     

Melanocyte differentiation antigen RAB38/NY-MEL-1 induces frequent antibody responses exclusively in melanoma patients

Expression pattern and immunogenicity are critical issues that define tumor antigens as diagnostic markers and potential targets for immunotherapy. The development of SEREX (serological analysis of recombinant expression libraries) has provided substantial progress in the identification of tumor antigens eliciting both cellular and humoral immune responses in cancer patients. By SEREX, we have previously identified RAB38/NY-MEL-1 as a melanocyte differentiation antigen that is highly expressed in normal melanocytes and melanoma tissues but not in other normal tissues or cancer types. In this study, we further demonstrate that RAB38/NY-MEL-1 is strongly immunogenic, leading to spontaneous antibody responses in a significant proportion of melanoma patients. The immune response occurs solely in malignant melanoma patients and was not detected in patients with other diseases, such as vitiligo, affecting melanocytes. Fine analysis of the spontaneous anti-RAB38/NY-MEL-1 antibody response reveals a polyclonal B cell recognition targeting various epitopes, although a dominant immunogenic region was preferentially recognized. Interestingly, our data indicate that this recognition is not rigid in the course of a patient’s response, as the dominant epitope changes during the disease evolution. Implications for the understanding of spontaneous humoral immune responses are discussed.Alfred Zippelius and Asma Gati contributed equally to this work.     

Broad antigenic coverage induced by vaccination with virus-based cDNA libraries cures established tumors

Effective cancer immunotherapy requires the release of a broad spectrum of tumor antigens in the context of potent immune activation. We show here that a cDNA library of normal tissue, expressed from a highly immunogenic viral platform, cures established tumors of the same histological type from which the cDNA library was derived. Immune escape occurred with suboptimal vaccination, but tumor cells that escaped the immune pressure were readily treated by second-line virus-based immunotherapy. This approach has several major advantages. Use of the cDNA library leads to presentation of a broad repertoire of (undefined) tumor-associated antigens, which reduces emergence of treatment-resistant variants and also permits rational, combined-modality approaches in the clinic. Finally, the viral vectors can be delivered systemically, without the need for tumor targeting, and are amenable to clinical-grade production. Therefore, virus-expressed cDNA libraries represent a novel paradigm for cancer treatment addressing many of the key issues that have undermined the efficacy of immuno- and virotherapy to date.