Serological analysis of recombinant cDNA expression libraries (SEREX) has led to the identification of several categories of new tumor antigens. We analyzed a testicular cDNA expression library with serum obtained from a breast cancer patient and isolated 13 genes designated NW-BR-1 through NW-BR-13. Of these, 3 showed tumor-restricted expression (NW-BR-1, -2 and -3), the others being expressed ubiquitously. NW-BR-3, representing 9 of 24 primary clones, showed tissue-restricted mRNA expression, being expressed in normal testis but not in 15 other normal tissues tested by Northern blotting. RT-PCR analysis showed strong NW-BR-3 expression in normal testis, weak expression in brain, kidney, trachea, uterus and normal prostate, and was negative in liver, heart, lung, colon, small intestine, bone marrow, breast, thymus, muscle, spleen, and stomach. NW-BR-3 mRNA expression was found in different tumor tissues and tumor cell lines by RT-PCR, thus showing a `cancer/testis´ (CT)-like mRNA expression pattern. NW-BR-3 shares 71% nucleotide and amino acid homology to a mouse gene cloned from mouse testicular tissue. Based on the mRNA expression pattern, NW-BR-3 represents a new candidate target gene for cancer immunotherapy. NW-BR-1 and NW-BR-2 also showed tumor-restricted mRNA expression. NW-BR-1 is a partial clone of the breast differentiation antigen NY-BR-1 previously identified by SEREX. NY-BR-1 is expressed in normal breast, testis and 80% of breast cancers. NW-BR-2 is identical to the CT antigen SCP-1, initially isolated by SEREX analysis of renal cancer. This study provides further evidence that SEREX is a powerful tool to identify new tumor antigens potentially relevant for immunotherapy approaches.
This article was published in Cancer Immunity, a Cancer Research Institute journal that ceased publication in 2013 and is now provided online in association with Cancer Immunology Research.
The search for breast cancer antigens that elicit humoral or cellular immune responses in breast cancer patients has a long history. There is early evidence for immune reactions against murine mammary tumor virus (1), and humoral immunity and delayed hypersensitivity against T/Tn antigens (2). More recent findings have shown antibody and T-cell responses to p53 (3) and HER-2/neu (4, 5). The main obstacle to identifying breast cancer antigens has been the difficulty in establishing breast cancer cell lines and T-cell lines required for T-cell epitope cloning, the approach successfully used to identify tumor antigens in melanoma. The introduction of the SEREX technique (6), a method to analyze the humoral immune response of cancer patients, has opened new perspectives in the field of tumor immunology. As SEREX does not rely on established tumor cell lines, this method is applicable to all types of cancer. Using the SEREX approach in breast cancer, several previously unknown breast cancer antigens have been identified and characterized (7, 8, 9, 10). In addition to using tumors as targets in SEREX analysis, screening of testicular cDNA expression libraries has led to the identification of new members of the cancer/testis antigen family, a family of tumor antigens expressed in normal testis and a variety of different tumor types (6, 7, 11). In the present study, a testis cDNA expression library was used as the antigen source for a SEREX analysis with serum derived from a breast cancer patient. In addition to cancer/testis antigen SCP-1 (7) and the breast differentiation antigen NY-BR-1 (8), this study has identified a new breast cancer antigen with tumor-restricted expression. The new antigen, referred to as NW-BR-3, has been characterized.
Identification of seroreactive clones
A total of 1.2 x 106 pfu from a testicular cDNA expression library were screened using serum from breast cancer patient NW1189 diluted 1:200. Twenty-four seroreactive clones were purified and sequenced. Comparison to GenBank and dbEST database entries revealed that these 24 clones were derived from 13 distinct genes, 12 known and 1 unknown, designated NW-BR-1 through NW-BR-13. Based on their expression profile in normal tissues and different tumor types, the identified gene products can be divided in two groups: one exhibiting tissue-restricted expression and one which is universally expressed. Thirteen clones representing three distinct genes belong to the first group: 3 NW-BR-1 clones, 1 NW-BR-2 clone, and 9 NW-BR-3 clones. The remaining 11 clones correspond to 9 known and one unknown genes, all universally expressed, based on the presence of ESTs in various normal tissues (Table 1).
NW-BR-1 encodes breast differentiation antigen NY-BR-1
Three clones (NW-BR-1) identified in this screening correspond to the NY-BR-1 gene. The NY-BR-1 cDNA clones isolated in this analysis are partial clones, the longest one extending from nucleotide 1754 to the 3'-end of the full length NY-BR-1 cDNA (nucleotide 4456).
NY-BR-1 was identified and characterized in a previous autologous breast cancer SEREX analysis (8). As shown previously, NY-BR-1 is expressed only in normal breast, normal testis, and approximately 80% of breast cancers, whereas it is not found in other tumor types. The expression profile for NY-BR-1 was confirmed by RT-PCR using additional breast cancer specimens (data not shown).
NW-BR-2 encodes cancer/testis antigen SCP-1
One clone (NW-BR-2) isolated in this screening was derived from SCP-1, a cancer/testis antigen previously identified by screening a testis cDNA expression library with serum from a patient with renal cancer (7). Comparison to dbEST and UniGene sequence entries shows SCP-1 to be expressed in normal testis and a variety of different tumors. The clone identified in this analysis contains the full length cDNA sequence and shows 100% cDNA identity to the SCP-1 GenBank entry (Accession No. X95654). This is the first time that SCP-1 has been isolated from a breast cancer SEREX analysis.
NW-BR-3 is the predominant antigen identified in this SEREX analysis
Nine of 24 clones isolated in this screening were derived from NY-BR-3. Comparison to GenBank showed an exact match to GenBank clone 18CGI1F2 derived from chromosome 18, a genomic clone which has not been characterized further. By comparing the NW-BR-3 cDNA sequence (Figure 1) to the genomic database, the exon-intron structure of this gene was deduced. As the NW-BR-3 gene consists of 8 exons, all RT-PCR primer-pairs used in the expression analysis (see below) were designed in different exons (trans-intronic). Comparison to dbEST entries showed a 99% match to a cDNA derived from a testicular cell line, and nearly exact matches to cDNAs derived from testis, retina, medulla, uterus carcinoma, anaplastic oligodendroglioma, and renal cancer (UniGene Cluster Hs.339651). None of the cDNAs found in the database permitted the extension of the NW-BR-3 cDNA sequence further 5' or 3'. The available NW-BR-3 cDNA is 1825 bp long, with a 1551 bp coding region, an 87 bp 5'-untranslated region, and a 187 bp 3'-untranslated region (excluding polyA). Because there was no stop codon identified in the putative 5'-untranslated region, 5'-RACE-PCR was performed to define the complete 5'-sequence using adaptor-ligated normal testis cDNA (Clontech). All RACE experiments confirmed that the isolated clone from the SEREX screening represents the full-length transcript. In addition, screening of the testicular library with a NW-BR-3 PCR-product probe isolated a single clone with identical cDNA sequence to NW-BR-3.
Northern Blot analysis using a NW-BR-3 PCR-product as probe showed a clear single signal in normal testis at approx. 1.9 kb (see Figure 3 below), also indicating that the NW-BR-3 cDNA sequence identified represents the full length NW-BR-3 transcript. The open reading frame of NW-BR-3 spanning nucleotides 88 to 1638 encodes a polypeptide of 516 amino acids with a molecular weight of 62 kDa.
Identification of mouse NY-BR-3 homolog
Databank analysis with the NW-BR-3 cDNA identified a mouse cDNA (GenBank Accession No. AK016821) derived from mouse testis which shows 71.4% homology at the nucleotide level, with 70.5% amino acid homology in the predicted protein sequences. Comparison of the coding region of the mouse homolog to the NW-BR-3 sequence places the translation initiation site of the mouse gene at position 346 of the NW-BR-3 cDNA sequence, demonstrating that the putative NW-BR-3 polypeptide is longer in the amino terminal region than the mouse homolog (Figure 2). Analysis of mouse dbEST database entries showed matches to cDNAs derived from testis, embryonic tissue and mammary gland. No information regarding the function of the mouse gene was available in the literature.
Expression profile of NW-BR-3
Database search with the NW-BR-3 cDNA sequence showed exact matches to cDNAs derived from testis, retina, and several different cancers (UniGene Cluster Hs.339651). Northern Blot analysis using commercially available panels of normal tissues (MNT-H1 and -H2, Clontech) and a NW-BR-3 PCR product probe showed a transcript of approx. 1.9 kb only in normal testis, but not in any of the other 15 normal tissues tested (Figure 3).
RT-PCR was performed to evaluate NW-BR-3 expression in a broader panel of different normal tissues, tumors and tumor cell lines. In contrast to the Northern Blot results, RT-PCR showed weak signals in brain, kidney, trachea, uterus and normal prostate, and a stronger signal in normal testis, but was negative in liver, heart, lung, colon, small intestine, bone marrow, breast, thymus, muscle, spleen, and stomach. As RT-PCR is known to be highly sensitive, the positive RT-PCR signals in brain, kidney, and prostate likely reflect a low level mRNA expression in those tissues not detectable by Northern Blot. In tumors and tumor cell lines, positive signals were seen in 2 of 13 breast cancers (weak), 4 of 5 renal cancers (moderate), 1 of 5 melanomas (weak), 1 of 4 prostate cancers (weak), 1 of 4 ovarian cancers (strong), 1 of 4 transitional cell carcinomas (weak), and in some tumor cell lines (Figure 4). The RT-PCR signals observed in tumor mRNA are in general weaker than the signal in testis; however, as RT-PCR is non-quantitative, the relative abundance of NW-BR-3 mRNA in testis, tumor cells, and in non-testicular normal tissues cannot be determined.
SEREX analysis of a range of different human tumor types has identified a number of tumor antigens with diagnostic and therapeutic potential (6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). Using expression libraries of testis rather than tumor as the antigen source has extended the range of defined antigens, particularly those with the characteristic features of CT antigens (7, 20). SEREX-defined tumor antigens have been classified into several categories, including CT antigens, differentiation antigens, mutational antigens, amplified/overexpressed antigens, splice variant antigens, and viral antigens (21). Antigens in each category are defined by either structural abnormalities or characteristic expression patterns. New cancer antigens are assigned to a category following careful analysis of structural abnormalities and the expression pattern in normal and tumor tissues by comparison to EST databases, Northern blotting or RT-PCR.
The present study extends our previous SEREX analysis of breast cancer (7, 8, 10). We have identified 24 seroreactive clones representing 13 distinct gene products. Comparison to database entries does not reveal any structural abnormalities in any of these clones. Three antigens (NW-BR-1, -2, and -3) showed tumor-restricted expression. Two of these antigens correspond to NY-BR-1 and SCP-1, which have previously been identified by SEREX (7, 8). NW-BR-3 is a new tumor antigen with a `cancer/testis´ -like pattern of expression.
NY-BR-1 was first identified in an autologous SEREX breast cancer screen (8). NY-BR-1 is a breast differentiation antigen with a highly restricted expression pattern; it is expressed in normal breast and normal testis but not in other normal tissues. Furthermore, while NY-BR-1 is expressed in 80% of breast cancers, tumors of other histological types are NY-BR-1 negative. Differentiation antigens, such as NY-BR-1, with a normal expression limited to non-vital organs, can be considered attractive vaccine targets. Melanocyte differentiation antigens, such as gp100, Melan-A, and tyrosinase, have been used in melanoma vaccines, and immune responses directed against these targets have led to tumor regression as well as vitiligo. We are currently evaluating the CD8+ T cell response to NY-BR-1 in breast cancer patients. In addition to its potential as a target antigen for immunotherapy, NY-BR-1 might be a useful diagnostic marker for breast cancer.
SCP-1 was previously identified by screening a testicular library with serum from a patient with renal cancer (12). SCP-1 is a component of the synaptonemal complex formed during meiotic prophase (22, 23). SCP-1 was shown to be expressed in one third of breast cancers and malignant gliomas by Tuereci et al. (7), as well as in about one fourth of gastric cancers (24). CT antigens represent ideal target molecules for cancer immunotherapy. Because of the lack of MHC class I expression in CT-positive germ cells in the testis, immune responses directed against CT antigens target tumor tissue selectively. In fact, no adverse effects have been seen in several immunization trials using CT antigen vaccines (25, 26, 27).
The predominant antigen detected in this screening was NW-BR-3, represented by 9 clones. A database search revealed sequence identity to a genomic clone derived from chromosome 18 in GenBank, and matches to cDNAs derived from normal testis, retina, medulla, and different tumors in the dbEST database. The expression pattern of this new antigen in normal tissues was confirmed by Northern blotting, with a signal detected only in normal testis. RT-PCR showed additional weak signals in normal brain, kidney and prostate. The discrepancy between Northern blotting and RT-PCR results is most likely due to the higher sensitivity of the RT-PCR technique. Analysis of the mRNA expression in a panel of different tumor tissues by RT-PCR showed NW-BR-3 to be expressed in some tumors and tumor cell lines. As RT-PCR is not a quantitative assay and mRNA levels do not necessarily parallel the level of protein expression, it is currently unclear whether the expression of NW-BR-3 protein in tumor is indeed higher than that in non-testicular normal tissues. The production of recombinant NW-BR-3 protein will facilitate the generation of monoclonal antibodies for immunohistochemical analysis of normal and malignant tissues, and this crucial issue can then be addressed.
Immunohistochemical assessment of tumor antigens frequently shows heterogeneous expression in a single tumor specimen, particularly in solid tumors (28, 29). Antigen-specific immunotherapy approaches for solid tumors will therefore have to be designed so as to target multiple tumor antigens at the same time to reduce the risk of immune escape of antigen-loss variant tumor cells. In this regard, the continued effort to identify new targets for immunotherapy in cancer is essential for the development of such polyvalent cancer vaccines. Our current study demonstrates the value of the SEREX technology in this continuing endeavor.
Materials and methods
Tumor tissues, cell lines and serum
Tumor tissues and cell lines were obtained and established at Krankenhaus Nordwest in Frankfurt.
The serum used in this SEREX study was obtained from a 60-year-old female patient treated at Krankenhaus Nordwest, Frankfurt, Germany. The patient was diagnosed with metastatic breast cancer 14 years after a mastectomy had been performed for a primary breast cancer (ductal carcinoma) involving lymph nodes and pleura. She received chemotherapy, with complete remission of all metastatic sites. One year later she developed lymph node metastases in the supraclavicular region, but again experienced a complete remission which lasted for 14 months following chemotherapy.
RNA extraction and construction of a cDNA expression library
Total RNA was extracted from normal testicular tissue by the conventional CsCl-guanidine thiocyanate method. A cDNA library was constructed in a lambda-ZAP Express vector, using a commercial cDNA library kit (Stratagene).
Immunoscreening of the cDNA library
The amplified cDNA expression library was screened with the breast cancer serum (NW1189) at a 1:200 dilution. The screening procedure was as described previously (9). Briefly, the serum was diluted 1:10, preabsorbed with phage-transfected Escherichia coli lysate, further diluted to 1:200, and incubated overnight at room temperature with the nitrocellulose membranes (Schleicher & Schüll) containing the phage plaques at a density of 4,000-5,000 pfu per 130 mm plate. After washing, the filters were incubated with alkaline phosphatase-conjugated goat anti-human Fcgamma secondary antibodies and the reactive phage plaques were visualized by incubating with 5-bromo-4-chloro-3-indolyl-phosphate and nitroblue tetrazolium.
Sequence analysis of the reactive clones
The reactive clones were subcloned and purified to pBS+ plasmid forms (Stratagene). Plasmid DNA was prepared by using the Wizard Miniprep DNA Purification System (Promega). The inserted DNA was evaluated by EcoRI-XhoI restriction mapping, and clones representing different cDNA inserts were sequenced. Sequencing was performed by the GATC Sequencing Service, Konstanz (Germany), using Applied Biosystems PRISM automated sequencers. DNA and amino acid sequences were compared with sequences in the GenBank and dbEST databases. Genes identical to entries in GenBank were classified as known genes, whereas those sharing sequence identity only to dbEST entries and those not matching entries in either GenBank or dbEST were classified as unknown genes.
To evaluate the mRNA expression pattern of the cloned cDNA in normal and malignant tissues, total RNA was extracted from tumor tissues and cell lines using the RNeasy kit (Qiagen), and normal tissue RNA was obtained commercially (Clontech). Gene-specific oligonucleotide primers, with an estimated primer melting temperature of 65-70°C, were designed to amplify cDNA segments 300-600 bp in length (refer to Figure 1 for primer sequences). All primers were synthesized commercially (Roth, Germany). RT-PCR was performed using 30 amplification cycles and an annealing temperature of 60°C in a thermal cycler (Eppendorf) and the products were analyzed by 1.3% gel electrophoresis and visualized with ethidium bromide.
Northern blot analysis
Northern blot analysis was performed using commercial poly(A)+ (2 µg/lane) Human Multiple Tissue Northern (MTN™) Blot I and II (Clontech). 32P-labeled PCR probes 300-600 bp in length were used, and blots were hybridized and washed according to the manufacturer's protocol (ExpressHyb kit, Clontech).
Rapid amplification of cDNA ends (RACE)
RACE reactions (5'- and 3'-RACE) were performed using gene-specific and adaptor-specific primers, Marathon-Ready normal testis cDNA and Advantage II Polymerase (Clontech). Products were ligated into the PCR-direct cloning vector pGEMT and analyzed by restriction mapping and sequencing.
Hybridization screening of a testicular library
A testicular cDNA expression library was screened using a NW-BR-3 PCR product as a probe (refer to Figure 1 for primer sequences), as described in the Stratagene manual. Briefly, a total of 5 x 104 pfu per 150 mm plate were transferred to nitrocellulose membranes (Schleicher & Schüll), the membranes were submerged in denaturation solution (1.5 M NaCl and 0.5 M NaOH) for 5 minutes, transferred into neutralization solution (1.5 M NaCl and 0.5 M Tris-HCl) for 5 minutes, and rinsed in 0.2 M Tris-HCl and 2 x SSC. The membranes were hybridized to a 32P-labeled DNA probe at high stringency (68°C, aqueous buffer) and washed at high stringency. Positive clones were subcloned and purified to pBS+ plasmid forms as described above.
This work was supported by the Cancer Research Institute/Avon Foundation Clinical Investigator Award.
- Received June 11, 2002.
- Accepted June 11, 2002.
- Copyright © 2002 by Dirk Jäger