Skip to main content
  • AACR Publications
    • Blood Cancer Discovery
    • Cancer Discovery
    • Cancer Epidemiology, Biomarkers & Prevention
    • Cancer Immunology Research
    • Cancer Prevention Research
    • Cancer Research
    • Clinical Cancer Research
    • Molecular Cancer Research
    • Molecular Cancer Therapeutics

AACR logo

  • Register
  • Log in
  • My Cart
Advertisement

Main menu

  • Home
  • About
    • The Journal
    • AACR Journals
    • Subscriptions
    • Permissions and Reprints
  • Articles
    • OnlineFirst
    • Current Issue
    • Past Issues
    • Meeting Abstracts
    • Cancer Immunology Essentials
    • Collections
      • COVID-19 & Cancer Resource Center
      • Toolbox: Coding and Computation
      • Toolbox: Signatures and Cells
      • "Best of" Collection
      • Editors' Picks
  • For Authors
    • Information for Authors
    • Author Services
    • Best of: Author Profiles
    • Submit
  • Alerts
    • Table of Contents
    • Editors' Picks
    • OnlineFirst
    • Citation
    • Author/Keyword
    • RSS Feeds
    • My Alert Summary & Preferences
  • News
    • Cancer Discovery News
  • COVID-19
  • Webinars
  • Search More

    Advanced Search

  • AACR Publications
    • Blood Cancer Discovery
    • Cancer Discovery
    • Cancer Epidemiology, Biomarkers & Prevention
    • Cancer Immunology Research
    • Cancer Prevention Research
    • Cancer Research
    • Clinical Cancer Research
    • Molecular Cancer Research
    • Molecular Cancer Therapeutics

User menu

  • Register
  • Log in
  • My Cart

Search

  • Advanced search
Cancer Immunology Research
Cancer Immunology Research
  • Home
  • About
    • The Journal
    • AACR Journals
    • Subscriptions
    • Permissions and Reprints
  • Articles
    • OnlineFirst
    • Current Issue
    • Past Issues
    • Meeting Abstracts
    • Cancer Immunology Essentials
    • Collections
      • COVID-19 & Cancer Resource Center
      • Toolbox: Coding and Computation
      • Toolbox: Signatures and Cells
      • "Best of" Collection
      • Editors' Picks
  • For Authors
    • Information for Authors
    • Author Services
    • Best of: Author Profiles
    • Submit
  • Alerts
    • Table of Contents
    • Editors' Picks
    • OnlineFirst
    • Citation
    • Author/Keyword
    • RSS Feeds
    • My Alert Summary & Preferences
  • News
    • Cancer Discovery News
  • COVID-19
  • Webinars
  • Search More

    Advanced Search

Articles

Humoral immune responses to testis antigens in sera from patients with prostate cancer

Luke H. Hoeppner, Jason A. Dubovsky, Edward J. Dunphy and Douglas G. McNeel
Luke H. Hoeppner
Department of Medicine, University of Wisconsin, Madison, WI 53792, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jason A. Dubovsky
Department of Medicine, University of Wisconsin, Madison, WI 53792, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Edward J. Dunphy
Department of Medicine, University of Wisconsin, Madison, WI 53792, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Douglas G. McNeel
Department of Medicine, University of Wisconsin, Madison, WI 53792, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI:  Published January 2006
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Abstract

Tumor vaccines represent one type of molecularly targeted therapy being investigated for the treatment of prostate cancer. Although many prostate-specific proteins are being tested as target antigens for prostate cancer vaccines, most are not natural targets of an immune response in patients with cancer. Using sera from cancer patients, several research groups have identified a large family of immunologically recognized proteins whose expression is normally confined to immune-privileged testis tissue but which may be expressed in cancers of different histological origins. These proteins, so-called cancer-testis (CT) antigens, are appealing targets for immune-based therapies because they are essentially tumor-restricted antigens and there is less risk of preexisting immune tolerance. In addition, specifically targeting these proteins by means of vaccines should reduce the risk of potential autoimmune reactions to normal tissues. In the current study, we hypothesize that prostate CT antigens can be identified using a SEREX screening method with sera from patients with prostate cancer and probing with a human testis cDNA expression library. We have identified several potential prostate cancer antigens with predominantly testis-specific expression in normal tissues, including MAD-CT-1 (protamine 2) and MAD-CT-2. Each was independently identified from different subjects with prostate cancer. Antigens identified by these studies can be investigated further as potential prostate cancer tumor antigens.

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.

Introduction

Prostate cancer is a significant health problem that is common in men above the age of 50. It is the most commonly diagnosed malignancy, and the second leading cause of cancer-related death, in men in the United States (1). At present, there is no cure for metastatic prostate cancer, and approximately 12% of patients diagnosed with prostate cancer ultimately die from the disease. There is considerable interest in developing molecularly targeted therapies for prostate cancer, including tumor vaccines (2). The results of several clinical vaccine trials suggest a clinical benefit to patients with prostate cancer, and phase III trials are currently underway in patients with androgen-independent prostate cancer (3, 4). The antigens chosen for inclusion in antigen-specific prostate cancer vaccines have predominantly been proteins with known prostate-restricted expression, including prostate-specific antigen, prostate-specific membrane antigen, and prostatic acid phosphatase. There is little evidence that any one of these antigens is superior to another as a vaccine target. To date, there has been little evaluation of prostate cancer-associated proteins that may be natural targets of CTLs. In contrast, the identification of antigens recognized by melanoma TILs has guided the evaluation of specific antigens for melanoma tumor vaccines (5, 6). Identifying immunologically recognized proteins in patients with prostate cancer could lead to the development of other potential vaccine target antigens. Unfortunately, the paucity of prostate cancer tissue obtained from individual subjects, and the difficulties associated with propagating autologous prostate cell lines compared with melanoma cell lines, has for the most part prohibited an evaluation of TILs analogous to those conducted with melanoma (7).

It has already been demonstrated that IgG responses to tumor-associated proteins exist in cancer patients, making it possible to use human sera to identify immunologically-recognized potential tumor antigens (8). SEREX (serological analysis of recombinant cDNA expression libraries of human tumors), is a robust methodology that uses IgG antibody-based screening to identify potential antigens of a tissue-specific cDNA expression library using human sera (8). Many antigens discovered through SEREX are known targets of CTLs (9, 10). Given the limitations described above, SEREX has become an attractive method for identifying tumor-associated antigens of solid tumors, including prostate cancer (11, 12, 13, 14, 15). The use of SEREX in other solid tumor systems has led to the identification of a large family of proteins whose expression is normally confined to testis tissue (16). Antigens restricted to the testis are considered immunologically privileged, given the absence of MHC molecule expression on germ cells (17). The aberrant expression in cancers of proteins that are normally restricted to germ cells may make them available for immune recognition (11). Members of this family of CT antigens, which includes MAGE, BAGE, GAGE-1, HOM-MEL-40, and NY-ESO-1, are expressed in a variety of solid tumors and have been found to be immunologically recognized in the sera of patients with cancers of diverse types (16, 18). Given their restricted tissue expression, in many cases, cancer-specific expression, CT antigens are promising candidates for antitumor vaccines (18). Moreover, in the case of prostate cancer, many patients with metastatic disease are treated with surgical castration. In this context, CT antigens expressed in prostate cancer cells should be unique tumor antigens.

Several members of the CT antigen family are expressed in prostate cancer tissues, including NY-ESO-1, LAGE-1, and XAGE-1 (19, 20, 21). This suggests that several known CT antigens warrant investigation as potential prostate tumor vaccine antigens. Other research groups have identified CT antigens in patients with breast cancer (22, 23) and with renal cell cancer (24) by using sera from patients to screen a testis cDNA expression library. To our knowledge, there have been no published reports of the direct identification of prostate CT antigens. In this report, we discuss whether prostate CT antigens can be identified prospectively using sera obtained from patients with prostate cancer. We used SEREX to screen for antigenic proteins, expressed from a human testis-tissue cDNA library, that were recognized by high-titer IgG antibodies in prostate cancer patients’ sera. We identified several potential prostate cancer antigens. Two of the proteins were identified independently in sera from separate individuals. These proteins were most highly expressed in testis tissue, but demonstrated low-level expression in some other, normal tissues. One of these, MAD-CT-2, was expressed in both normal and malignant prostate tissues. Expression of MAD-CT-1 was not detectable in normal prostate tissue, but was detectable in 2 of 10 metastatic prostate cancer specimens.

Results

Testis protein-specific IgG in the sera of patients with prostate cancer

To identify possible CT antigens from patients with prostate cancer, we used the SEREX methodology to screen a normal testis cDNA phage expression library with sera from prostate cancer patients. Because the identification of antigens is essentially dependent on the choice of sera for screening, this analysis was conducted with sera from prostate cancer patients from a variety of different clinical scenarios. Many of these patients had atypical histories of prostate cancer regression without specific treatment or had evidence of antibody responses to other prostate cancer antigens (25), as described in Table 1. SEREX screening was conducted with paired sera to speed the analysis, and immunoreactive plaques were isolated and rescreened individually with the unpaired sera used for initial detection. A strongly immunoreactive plaque identified from this initial screen is shown in Figure 1. From this process, 122 potentially immunoreactive phage plaques were identified. These phages were then rescreened with the reactive sera at lower plaque density to isolate purified phages and to eliminate false positives and phages with weak immunoreactivity from further evaluation. Following this, 27 immunoreactive phages were identified and ultimately sequenced. Seven of the twenty-seven sequenced phages were found to be unique (shown in Table 2). It is noteworthy that two of these phages, encoding MAD-CT-1 or MAD-CT-2, were each isolated from the sera of two different patients.

View this table:
  • View inline
  • View popup
Table 1

Sera from subjects with prostate cancer used for SEREX analysis.

Figure 1
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 1

Testis protein-specific IgG can be detected in the sera of patients with prostate cancer. A λ phage cDNA expression library constructed from normal human testis tissue was used to transduce E. coli for SEREX immunoscreening. A representative primary screen used to identify immunoreactive phage plaques is shown. The box shows a magnification of the area surrounding an immunoreactive plaque.

View this table:
  • View inline
  • View popup
Table 2

Testis proteins identified by SEREX using sera from patients with prostate cancer.

Antigen-specific IgG in the sera of other patients with prostate cancer

To determine if any of the 7 gene products identified and shown in Table 2 were recognized by IgG in sera from other patients with prostate cancer, a larger analysis was performed with phage immunoscreening. For this experiment, purified phages encoding each antigen were directly spotted to bacterial lawns in multiple replicates and evaluated using sera from 109 patients in various stages of prostate cancer and from 52 male control blood donors without histories of prostate cancer. Examples are shown in Figure 2, and the findings are summarized in Table 3. None of the antigens were recognized significantly more frequently in the sera of patients versus controls (P > 0.05). Consequently, in order to prioritize these proteins for further evaluation, the three antigens recognized by the greatest number of patients relative to controls, MAD-CT-1, MAD-CT-2, and MAD-CT5, were chosen.

Figure 2
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 2

Antigen-specific IgG can be detected in the sera of other patients with prostate cancer. A schematic layout of the antigen-encoding phages (panel A) and data from three separate subjects with prostate cancer (panels B-D) are shown. "POS IgG" indicates a positive control phage encoding human IgG, and "NEG" indicates a negative control phage not encoding an immunoreactive protein. The arrows point to the immunoreactive proteins identified.

View this table:
  • View inline
  • View popup
Table 3

Antigen-specific IgG detected in sera from patients with prostate cancer and male control blood donors.

MAD-CT-5-specific IgG antibodies

The protein encoded by MAD-CT-5, the Sjögren’s syndrome B antigen (SSB, autoantigen La), is a ubiquitously expressed protein associated with RNA polymerase III (26, 27). It is also a known autoantigen typically recognized in patients with Sjögren’s syndrome. A commercially available ELISA method was used to assay for the presence of MAD-CT-5 (SSB)-specific antibodies (Figure 3A). This confirmed the presence of MAD-CT-5-specific antibodies in the sera of patient # 20, from which the antigen was identified, thereby validating our SEREX immunoscreening methodology. This ELISA methodology was used in a larger panel consisting of normal controls (n = 47) and subjects with prostate cancer (n = 95) to determine if low-titer antibodies to SSB can be detected, perhaps below the level of detection of our immunoblot methodology. As shown in Figure 3B, antibody responses were detectable in a few individuals, consistent with our phage immunoscreening findings. IgG responses to SSB, however, were not detected more frequently in the sera of patients with early (n = 37) or late stage (n = 58) prostate cancer as compared with control male blood donors (n = 47).

Figure 3
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 3

MAD-CT-5-specific IgG antibodies can be detected by ELISA. (A) IgG antibodies specific for the SSB antigen (MAD-CT-5) were analyzed by ELISA (Kallestad Anti-La Test Kit, Bio-Rad). The colorimetric ELISA is shown. A1, B1: Negative controls. C1, D2: 0 U/mL. E1, F1: 2 U/mL. G1, H1: 8 U/mL. A2, B2: 30 U/mL. C2, D2: 100 U/mL standards. E2, F2: Positive control sera. A3-H4: Sera from subjects without MAD-CT-5 immunoreactivity. A5, B5: Sera from subject # 20, from which MAD-CT-5 was originally identified. (B) ELISA was conducted with sera obtained from 47 male control blood donors, 37 subjects with early-stage (nonmetastatic) prostate cancer, and 58 subjects with metastatic prostate cancer. The SSB-specific IgG in U/mL is shown, determined by comparison with a standardized control. The line represents the mean +3 SD of data from the normal control population, above which results were classified as positive.

MAD-CT-1 and MAD-CT-2 expression

MAD-CT-1 (protamine-2) is a known spermatid protein with essentially testis-specific tissue expression (28), whereas expression of MAD-CT-2 has not been reported previously. In order to determine the expression of MAD-CT-2 in normal tissues, PCR was performed with cDNA from several normal human tissues (Figure 4A). As is shown, expression of MAD-CT-1 and MAD-CT-2 was highest in normal testis tissue. MAD-CT-1 expression was also detectable in brain tissue, and lower expression was detectable in bladder tissue. MAD-CT-2 was also found to have low-level expression in normal bladder and prostate tissue. Similar results were found by RT-PCR using total tissue RNA (data not shown). To determine if either of these antigens might be expressed in prostate cancer, a similar analysis was conducted with cDNA specimens obtained from 10 separate prostate cancer metastases (Figure 4B). By this analysis, we found low but detectable levels of MAD-CT-1 transcript in 2 of 10 prostate cancer specimens (patients # 8 and # 9, Figure 4B). MAD-CT-2 transcript was detectable in 9 of the 10 prostate cancer specimens.

Figure 4
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 4

MAD-CT-1 and MAD-CT-2 are predominantly expressed in testis and are expressed in some prostate cancer tissues. (A) Gene-specific primers for MAD-CT-1, MAD-CT-2, or actin were used to detect transcript in cDNA samples obtained from normal human liver, lung, testis, colon, bladder, prostate, heart, brain, kidney, placenta, skeletal muscle, spleen or thymus (Biochain, Hayward, CA, USA). (B) Gene-specific primers were used to amplify MAD-CT-1, MAD-CT-2, or actin from cDNA specimens derived from metastatic prostate cancer tissue specimens (n = 10). A plus sign indicates a positive control using plasmid DNA encoding either MAD-CT-1 or MAD-CT-2 as template DNA. A minus sign indicates a negative control using nonspecific plasmid DNA as template DNA.

Discussion

The group of antigens known as CT antigens, or germ cell antigens, is a large family of proteins expressed primarily in adult, male germ cell tissues. These proteins are also expressed in cancers of different origins. It is believed that these antigens may escape normal immune recognition, given their restricted expression in MHC class I-negative germ cells (29). Thus, there has been considerable interest in identifying CT antigens, as they may serve essentially as tumor-restricted antigens for the development of vaccines that are at lower risk for preexisting immune tolerance and for creating potential autoimmune reactions to normal tissues. Several members of the CT antigen family are expressed in prostate cancer, including PAGE-4 (30), NY-ESO-1 (19), LAGE-1 (21), XAGE-1 (31), and members of the MAGE-A family (32). To date, we are unaware of any published reports that directly identify prostate cancer CT antigens using sera from patients with prostate cancer. In this study, we used SEREX to screen a normal testis cDNA library using sera from patients with prostate cancer, including sera from patients with early stage, nonmetastatic prostate cancer (n = 10) as well as from patients with metastatic prostate cancer (n = 12). Within this population, we included sera from patients with clinical histories suggesting disease response or with previous evidence of immune response to other prostate cancer antigens. We identified several proteins by this method, of which MAD-CT-1 (PRM2) and MAD-CT-2 are likely prostate cancer antigens. Their expression, however, is not entirely germ cell-specific, as has been reported for some CT antigens such as NY-ESO-1 (11).

Northern blot analysis and immunohistochemical analysis (28), as well as evaluation of EST databases (33, 34), had previously suggested that, of the 7 proteins found in this study, PRM2 is testis-specific in terms of tissue expression. It had also been previously identified as an autoantigen in infertile men without cancer (35) and in patients with sarcoma (36). Curiously, in the latter study the authors suggested that the protein is ubiquitously expressed based on their quantitative RT-PCR analysis, and they did not explore its potential as a CT antigen (36). We suspect that the primers used in their study were not entirely specific for PRM2, as we have performed similar quantitative RT-PCR analysis demonstrating essentially tissue-specific expression (data not shown). Our data are also in concordance with the gene expression predicted by EST analysis (37), in which expression was predicted to be predominantly in testis, but with low-level expression in brain and placenta (34). The identification of PRM2 from a sarcoma library, and its detection in prostate cancer but not normal prostate tissue, suggests that it is indeed a tumor antigen. Furthermore, the earlier identification of PRM2 as an autoantigen in infertile men, as well as in subjects with various cancers, also suggests that it is an immunogenic protein. Strictly speaking, however, the detection of PRM2 expression in other normal tissues, including brain, suggests it is not a "pure" CT antigen.

Little is known of the gene product encoded by MAD-CT-2, and it has not been previously named or characterized. The cDNA had been identified earlier by sequencing a testis cDNA library, but at present there is no other information about tissue expression, nor is there any information in the NIH EST database (34). From our RT-PCR analysis of RNA from normal human tissues, it does appear to be predominantly expressed in testis, with lower levels of expression in normal bladder and prostate tissues. The predicted gene product has a predominance of charged residues (38.5% aspartate, glutamate, lysine, or arginine residues), and 24.2% of the amino acids are glycine residues. The presence of many charged residues, and the fact that it exhibits some homology to known DNA-binding proteins, suggest that it may be a DNA-binding protein. It also, however, demonstrates homology to several members of a large family of Golgi proteins, many of which are being characterized as autoantigens (38). The testis-predominant expression of MAD-CT-2, its expression in prostate cancer tissues, and the presence of antibodies to this protein in the sera of several patients with prostate cancer, suggest that it is also a prostate cancer antigen. Like MAD-CT-1, however, it cannot be classified as a "pure" CT antigen, given that its expression is not entirely restricted to germ cells and tumor cells. Given its restricted expression to testis, bladder, and prostate, however, this gene product could potentially be investigated as a prostate or bladder tumor antigen.

The autoantigen La (MAD-CT-5) was recognized in the sera of one individual. Complete medical histories were not available for the patients from whom the sera was taken, thus we do not know whether this patient had symptoms of Sjögren’s syndrome. The known, generally ubiquitous, expression of this protein suggests that it is not a CT antigen. The identification of this protein in this study, however, did permit us to validate our SEREX methodology using clinically standardized reagents in an ELISA. As indicated, we saw few significant IgG responses to this protein by immunoscreening with phage or by ELISA in a larger panel of patients versus controls.

The other proteins identified have not specifically been identified as autoantigens in other studies, to our knowledge. There have, however, been multiple reports of autoantibody responses to other ribosomal proteins, similar to MAD-CT-7, in other cancers and autoimmune diseases, suggesting that these are common autoantigens (39, 40). Curiously, we did not identify other commonly recognized CT antigens such as NY-ESO-1, LAGE-1, or members of the MAGE-A family. In particular, we expected to detect NY-ESO-1 in our analysis, as a report by Nakada and colleagues suggested that NY-ESO-1 is expressed in 38% of prostate tumor specimens (19). The detection of antibodies to a particular protein in our analysis, however, was strictly dependent on the choice of sera used. We specifically chose sera for our primary SEREX screening to include sera from patients with early stage, nonmetastatic prostate cancer, as well as patients with metastatic prostate cancer, for the broadest possible sampling. In the earlier report of NY-ESO-1 expression in prostate cancer, antibodies to NY-ESO-1 were only detectable in the sera of 8% of patients with advanced-stage prostate cancer (19). Consequently, our population of sera samples for screening, of which only 12 had metastatic disease, might not have been large enough to identify NY-ESO-1 in particular. In related studies, we are currently assembling a large panel of phages encoding previously identified CT antigens in order to identify immunologically recognized CT antigens by a similar methodology, using sera from many patients with prostate cancer. Together with the current study, these studies should further define known CT antigens that could be explored as therapeutic or diagnostic target antigens for prostate cancer.

Materials and methods

Subject population

Sera were obtained from patients with various stages of prostate cancer who gave written Internal Review Board-approved informed consent for their blood products to be used for immunological research. Blood was collected at the University of Washington Medical Center (Seattle, WA, USA) between 1997 and 2001, and at the University of Wisconsin Hospital and Clinics (Madison, WI, USA) between 2001 and 2004. Sera were stored in aliquots at -80˚C until used. Control sera were obtained from age-matched male volunteer blood donors who also gave written informed consent.

SEREX

SEREX analysis was conducted in a manner similar to what we described previously (14). A phage cDNA expression library made from normal human testis tissue mRNA (TriplEx, BD Biosciences Clontech, Palo Alto, CA, USA) was used to transduce the E. coli strain XL-1 Blue (Stratagene, La Jolla, CA, USA). Transduced bacteria were then plated in top agarose in a lawn at a phage density of approximately 6500 pfu per 150-mm LB agar plate. After plaques developed, nitrocellulose filters (Millipore, Bedford, MA, USA) presoaked with 10 mM IPTG were overlaid to induce expression of recombinant proteins. After 12-16 h, the membranes were removed, washed twice with 10 mM Tris 8.0, 150 mM NaCl, 0.5% Tween-20 (TBST), and once with 10 mM Tris 8.0, 150 mM NaCl (TBS), blocked with TBST plus 1% BSA, and then probed with human sera diluted 1:100 in TBST plus 1% BSA overnight. Sera from 22 prostate cancer patients were arbitrarily paired to speed screening, and a mixture of 2 sera was used in each of 11 screens. Membranes were then washed and blocked again, and human IgG was detected with an alkaline phosphatase-conjugated monoclonal antihuman IgG antiserum (Sigma, St. Louis, MO, USA) diluted in TBST plus 1% BSA. After repeated washing, the membranes were developed with 0.3 mg/mL nitro blue tetrazolium chloride (NBT) (Fisher Biotech, Pittsburgh, PA, USA) and 0.15 mg/mL 5-bromo 4-chloro 3-indoylphosphate (BCIP) (Fisher Biotech) in 100 mM Tris 9.5, 100 mM NaCl, 5mM MgCl2. Immunoreactive plaques were then removed from the corresponding agar plates and the phages were eluted overnight in SM buffer (100 mM NaCl, 10 mM MgSO4, 35 µM Tris 7.5, and 0.01% gelatin). This process was repeated with multiple agar plates, such that 75,000 to 250,000 pfu were ultimately screened with each sera pair. To eliminate false positives prior to plaque purification, an aliquot of each eluted heterogeneous plaque was individually spotted in replicate fashion onto lawns of E. coli growing on LB agar plates. Plaques were allowed to establish as before, and were then transferred to IPTG-impregnated nitrocellulose membranes. The membranes were then probed individually with each of the two sera of the original pairing to determine which of the two sera contained phage-specific IgG. Plaques were then rescued to pBluescript-derived phagemids by transduction of the recombinase-positive E. coli strain BM25.8 (BD Biosciences Clontech, Palo Alto, CA, USA). Recombinant bacteria were selected on ampicillin-containing media, and purified plasmids were then sequenced using plasmid-specific primers and ABI BigDye terminator sequencing reactions (Perkin-Elmer, Foster City, CA, USA). The GenBank database was searched to identify the gene products encoded by the recombinant phagemids. Gene products were labeled according to the standard SEREX database nomenclature (for example, MAD for "Madison," CT for "cancer-testis").

Immunoblot screening

Purified phages corresponding to each of the sequenced plasmids were analyzed for immunoreactivity using a panel of sera from 109 prostate cancer patients and 52 normal controls. Lawns of XL-1 Blue E. coli were grown on LB agar plates, and purified plaques were individually spotted onto the lawns in replicate fashion. A phage encoding human IgG was included on each membrane as a positive control, and irrelevant purified phages were included on each membrane as negative controls. Protein expression was induced with IPTG, and phages were transferred to nitrocellulose membranes, as described above. Serum from each of the subjects and controls was used to screen the membranes. Immunoreactive plaques were recorded for each of the subjects, and the controls were observed and compared to the internal positive and negative control plaques. Four "blinded" individuals judged the immunoreactivity of plaques, and those plaques determined to be positive by at least three individuals were recorded as immunoreactive with the given sera, to reduce the subjectivity of some weakly immunoreactive plaques. A chi-square analysis was used to compare the number of immunoreactive plaques in subject and control populations.

MAD-CT-5 ELISA

Anti-SSB antibodies were detected using a commercial kit (Kallestad Anti-La Test Kit, Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. Sera were diluted 1:100 in sample diluent prior to analysis. IgG reactivity is reported in units per mL by comparison with a standardized reference.

Tissue expression analysis by PCR

Gene-specific oligonucleotide primers (Proligo, Boulder, CO, USA) were designed to amplify MAD-CT-1 (Forward: 5’-GGGGTACCAATGGTCCGATACCGCGTGAGGAG-3’, Reverse: 5’-GGAATTCTTAGTGCCTTCTGCATGTTCTCTT-3’), MAD-CT-2 (Forward: 5’-GAGGATATGAGATCAGAAAGA-3’, Reverse: 5’-TCGGTAGAAGAATGGGATGC-3’), and actin (Forward: 5’-TCATGAAGTGTGACGTTGACATCCGT-3’, Reverse: 5’-CTTAGAAGCATTTGCGGTGCACGATG-3’) from cDNA specimens by PCR. PCR cycling and detection was conducted in a MyCycler thermocycler (Bio-Rad Laboratories) with 35 cycles of amplification under the following conditions: 60 s at 94˚C, 60 s at annealing temperature (65˚C for MAD-CT-1, 58.7˚C for MAD-CT-2, or 55˚C for actin), and 3 min at 72˚C. Plasmid template DNA encoding MAD-CT-1 or MAD-CT-2 was used as a positive control. In preliminary experiments with serial dilutions of plasmid DNA, the threshold level of detection was found to be less than or equal to 48 copies of MAD-CT-1 template and less than or equal to 880 copies of MAD-CT-2 template. Template DNAs for this analysis included cDNA specimens from normal human tissues (Biochain, Hayward, CA, USA), and cDNA derived from 10 metastatic prostate specimens (5 soft tissue or lymph node metastases and 5 bone metastases) from 10 separate patients. The latter cDNA specimens were graciously provided by the laboratory of Dr. Robert Vessella (University of Washington, Seattle, WA, USA).

Acknowledgments

This work is supported by NIH (K23 RR16489), the Howard Hughes Medical Institute at the University of Wisconsin, and the Prostate Cancer Foundation (CaPCURE).

  • Received July 6, 2005.
  • Accepted December 1, 2005.
  • Copyright © 2006 by Douglas McNeel

References

  1. 1.↵
    1. Jemal A,
    2. Murray T,
    3. Ward E,
    4. Samuels A,
    5. Tiwari RC,
    6. Ghafoor A,
    7. Feuer EJ,
    8. Thun MJ
    . Cancer statistics, 2005. CA Cancer J Clin 2005;55:10–30.pmid:15661684
    OpenUrlCrossRefPubMed
  2. 2.↵
    1. McNeel DG,
    2. Malkovsky M
    . Immune-based therapies for prostate cancer. Immunol Lett 2005;96:3–9.pmid:15585302
    OpenUrlCrossRefPubMed
  3. 3.↵
    1. Small EJ,
    2. Rini B,
    3. Higano C,
    4. Redfern C,
    5. Nemunaitis J,
    6. Valone F,
    7. Kylstra J,
    8. Schellhammer PF
    . A randomized, placebo-controlled phase III trial of APC8015 in patients with androgen-independent prostate cancer (AiPCa) Proc Amer Soc Clin Oncol 2003; 22: 382. (abstract # 1534) 
  4. 4.↵
    1. Small EJ,
    2. Higano C,
    3. Smith D,
    4. Corman J,
    5. Centeno A,
    6. Steidle C,
    7. Gittelman M,
    8. Hudes G,
    9. Sacks N,
    10. Simons JW
    . A phase 2 study of an allogeneic GM-CSF gene-transduced prostate cancer cell line vaccine in patients with metastatic hormone-refractory prostate cancer (HRPC) ASCO Prostate Cancer Symposium 2005; abstract # 280. 
  5. 5.↵
    1. van der Bruggen P,
    2. Traversari C,
    3. Chomez P,
    4. Lurquin C,
    5. De Plaen E,
    6. Van den Eynde B,
    7. Knuth A,
    8. Boon T
    . A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 1991;254:1643–1647.pmid:1840703
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    1. Boon T
    . Tumor antigens recognized by cytolytic T lymphocytes: present perspectives for specific immunotherapy. Int J Cancer 1993;54:177–180.pmid:8486420
    OpenUrlCrossRefPubMed
  7. 7.↵
    1. Housseau F,
    2. Bright RK,
    3. Simonis T,
    4. Nishimura MI,
    5. Topalian SL
    . Recognition of a shared human prostate cancer-associated antigen by nonclassical MHC-restricted CD8+ T cells. J Immunol 1999;163:6330–6337.pmid:10570328
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    1. Sahin U,
    2. Tureci O,
    3. Schmitt H,
    4. Cochlovius B,
    5. Johannes T,
    6. Schmits R,
    7. Stenner F,
    8. Luo G,
    9. Schobert I,
    10. Pfreundschuh M
    . Human neoplasms elicit multiple specific immune responses in the autologous host. Proc Natl Acad Sci U S A 1995;92:11810–11813.pmid:8524854
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    1. Jäger E,
    2. Chen YT,
    3. Drijfhout JW,
    4. Karbach J,
    5. Ringhoffer M,
    6. Jager D,
    7. Arand M,
    8. Wada H,
    9. Noguchi Y,
    10. Stockert E,
    11. Old LJ,
    12. Knuth A
    . Simultaneous humoral and cellular immune response against cancer-testis antigen NY-ESO-1: definition of human histocompatibility leukocyte antigen (HLA)-A2-binding peptide epitopes. J Exp Med 1998;187:265–270.pmid:9432985
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. Greiner J,
    2. Ringhoffer M,
    3. Simikopinko O,
    4. Szmaragowska A,
    5. Huebsch S,
    6. Maurer U,
    7. Bergmann L,
    8. Schmitt M
    . Simultaneous expression of different immunogenic antigens in acute myeloid leukemia. Exp Hematol 2000;28:1413–1422.pmid:11146163
    OpenUrlCrossRefPubMed
  11. 11.↵
    1. Chen YT,
    2. Scanlan MJ,
    3. Sahin U,
    4. Tureci O,
    5. Gure AO,
    6. Tsang S,
    7. Williamson B,
    8. Stockert E,
    9. Pfreundschuh M,
    10. Old LJ
    . A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening. Proc Natl Acad Sci U S A 1997;94:1914–1918.pmid:9050879
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    1. McNeel DG
    . Prostate cancer antigens and vaccines, pre-clinical developments. In: Cancer Chemotherapy and Biological Response Modifiers, Annual 22. Giaccone G, Schilsky R, Sondel P. (Eds.) Oxford, England: Elsevier Limited; 2005; in press. 
  13. 13.↵
    1. Fossa A,
    2. Siebert R,
    3. Aasheim HC,
    4. Maelandsmo GM,
    5. Berner A,
    6. Fossa SD,
    7. Paus E,
    8. Smeland EB,
    9. Gaudernack G
    . Identification of nucleolar protein No55 as a tumour-associated autoantigen in patients with prostate cancer. Br J Cancer 2000;83:743–749.pmid:10952778
    OpenUrlCrossRefPubMed
  14. 14.↵
    1. Dunphy EJ,
    2. Eickhoff JC,
    3. Muller CH,
    4. Berger RE,
    5. McNeel DG
    . Identification of antigen-specific IgG in sera from patients with chronic prostatitis. J Clin Immunol 2004;24:492–501.pmid:15359108
    OpenUrlCrossRefPubMed
  15. 15.↵
    1. Dunphy EJ,
    2. McNeel DG
    . Antigen-specific IgG elicited in subjects with prostate cancer treated with flt3 ligand. J Immunother 2005;28:268–275.pmid:15838384
    OpenUrlPubMed
  16. 16.↵
    1. Scanlan MJ,
    2. Simpson AJ,
    3. Old LJ
    . The cancer/testis genes: review, standardization, and commentary. Cancer Immun 2004; 4: 1. [PubMed] 
  17. 17.↵
    1. Kowalik I,
    2. Kurpisz M,
    3. Jakubowiak A,
    4. Janecki A,
    5. Lukaszyk A,
    6. Szymczynski G
    . Evaluation of HLA expression on gametogenic cells isolated from human testis. Andrologia 1989;21:237–243.pmid:2774218
    OpenUrlPubMed
  18. 18.↵
    1. Kirkin AF,
    2. Dzhandzhugazyan KN,
    3. Zeuthen J
    . Cancer/testis antigens: structural and immunobiological properties. Cancer Invest 2002;20:222–236.pmid:11901543
    OpenUrlCrossRefPubMed
  19. 19.↵
    1. Nakada T,
    2. Noguchi Y,
    3. Satoh S,
    4. Ono T,
    5. Saika T,
    6. Kurashige T,
    7. Gnjatic S,
    8. Ritter G,
    9. Chen YT,
    10. Stockert E,
    11. Nasu Y,
    12. Tsushima T,
    13. Kumon H,
    14. Old LJ,
    15. Nakayama E
    . NY-ESO-1 mRNA expression and immunogenicity in advanced prostate cancer. Cancer Immun 2003; 3: 10. [PubMed] 
  20. 20.↵
    1. Fossa A,
    2. Alsoe L,
    3. Crameri R,
    4. Funderud S,
    5. Gaudernack G,
    6. Smeland EB
    . Serological cloning of cancer/testis antigens expressed in prostate cancer using cDNA phage surface display. Cancer Immunol Immunother 2004;53:431–438.pmid:14747957
    OpenUrlCrossRefPubMed
  21. 21.↵
    1. Lethe B,
    2. Lucas S,
    3. Michaux L,
    4. De Smet C,
    5. Godelaine D,
    6. Serrano A,
    7. De Plaen E,
    8. Boon T
    . LAGE-1, a new gene with tumor specificity. Int J Cancer 1998;76:903–908.pmid:9626360
    OpenUrlCrossRefPubMed
  22. 22.↵
    1. Jager D,
    2. Unkelbach M,
    3. Frei C,
    4. Bert F,
    5. Scanlan MJ,
    6. Jager E,
    7. Old LJ,
    8. Chen YT,
    9. Knuth A
    . Identification of tumor-restricted antigens NY-BR-1, SCP-1, and a new cancer/testis-like antigen NW-BR-3 by serological screening of a testicular library with breast cancer serum. Cancer Immun 2002; 2: 5. [PubMed] 
  23. 23.↵
    1. Jager D,
    2. Stockert E,
    3. Scanlan MJ,
    4. Gure AO,
    5. Jager E,
    6. Knuth A,
    7. Old LJ,
    8. Chen YT
    . Cancer-testis antigens and ING1 tumor suppressor gene product are breast cancer antigens: characterization of tissue-specific ING1 transcripts and a homologue gene. Cancer Res 1999;59:6197–6204.pmid:10626813
    OpenUrlAbstract/FREE Full Text
  24. 24.↵
    1. Tureci O,
    2. Sahin U,
    3. Zwick C,
    4. Koslowski M,
    5. Seitz G,
    6. Pfreundschuh M
    . Identification of a meiosis-specific protein as a member of the class of cancer/testis antigens. Proc Natl Acad Sci U S A 1998;95:5211–5216.pmid:9560255
    OpenUrlAbstract/FREE Full Text
  25. 25.↵
    1. McNeel DG,
    2. Nguyen LD,
    3. Storer BE,
    4. Vessella R,
    5. Lange PH,
    6. Disis ML
    . Antibody immunity to prostate cancer-associated antigens can be detected in the serum of patients with prostate cancer. J Urol 2000;164:1825–1829.pmid:11025777
    OpenUrlCrossRefPubMed
  26. 26.↵
    1. Chambers JC,
    2. Keene JD
    . Isolation and analysis of cDNA clones expressing human lupus La antigen. Proc Natl Acad Sci U S A 1985;82:2115–2119.pmid:3856888
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    1. Sturgess AD,
    2. Peterson MG,
    3. McNeilage LJ,
    4. Whittingham S,
    5. Coppel RL
    . Characteristics and epitope mapping of a cloned human autoantigen La. J Immunol 1988;140:3212–3218.pmid:2452201
    OpenUrlAbstract
  28. 28.↵
    1. Domenjoud L,
    2. Kremling H,
    3. Burfeind P,
    4. Maier WM,
    5. Engel W
    . On the expression of protamine genes in the testis of man and other mammals. Andrologia 1991;23:333–337.pmid:1724877
    OpenUrlPubMed
  29. 29.↵
    1. Uyttenhove C,
    2. Godfraind C,
    3. Lethe B,
    4. Amar-Costesec A,
    5. Renauld JC,
    6. Gajewski TF,
    7. Duffour MT,
    8. Warnier G,
    9. Boon T,
    10. Van den Eynde BJ
    . The expression of mouse gene P1A in testis does not prevent safe induction of cytolytic T cells against a P1A-encoded tumor antigen. Int J Cancer 1997;70:349–356.pmid:9033639
    OpenUrlCrossRefPubMed
  30. 30.↵
    1. Prikler L,
    2. Scandella E,
    3. Men Y,
    4. Engeler DS,
    5. Diener PA,
    6. Gillessen S,
    7. Ludewig B,
    8. Schmid HP
    . Adaptive immunotherapy of the advanced prostate cancer - cancer testis antigen (CTA) as possible target antigens. Aktuelle Urol 2004;35:326–330.pmid:15459874
    OpenUrlPubMed
  31. 31.↵
    1. Egland KA,
    2. Kumar V,
    3. Duray P,
    4. Pastan I
    . Characterization of overlapping XAGE-1 transcripts encoding a cancer testis antigen expressed in lung, breast, and other types of cancers. Mol Cancer Ther 2002;1:441–450.pmid:12479262
    OpenUrlAbstract/FREE Full Text
  32. 32.↵
    1. Kufer P,
    2. Zippelius A,
    3. Lutterbuse R,
    4. Mecklenburg I,
    5. Enzmann T,
    6. Montag A,
    7. Weckermann D,
    8. Passlick B,
    9. Prang N,
    10. Reichardt P,
    11. Dugas M,
    12. Kollermann MW,
    13. Pantel K,
    14. Riethmuller G
    . Heterogeneous expression of MAGE-A genes in occult disseminated tumor cells: a novel multimarker reverse transcription-polymerase chain reaction for diagnosis of micrometastatic disease. Cancer Res 2002;62:251–261.pmid:11782385
    OpenUrlAbstract/FREE Full Text
  33. 33.↵
    1. Schuler GD
    . Pieces of the puzzle: expressed sequence tags and the catalog of human genes. J Mol Med 1997;75:694–698.pmid:9382993
    OpenUrlCrossRefPubMed
  34. 34.↵
    1. Wheeler DL,
    2. Church DM,
    3. Edgar R,
    4. Federhen S,
    5. Helmberg W,
    6. Madden TL,
    7. Pontius JU,
    8. Schuler GD,
    9. Schriml LM,
    10. Sequeira E,
    11. Suzek TO,
    12. Tatusova TA,
    13. Wagner L
    . Database resources of the National Center for Biotechnology Information: update. Nucleic Acids Res 2004; 32: D35-D40. [PubMed] 
  35. 35.↵
    1. Samuel T,
    2. Kolk AH,
    3. Rumke P
    . Studies on the immunogenicity of protamines in humans and experimental animals by means of a micro-complement fixation test. Clin Exp Immunol 1978;33:252–260.pmid:102475
    OpenUrlPubMed
  36. 36.↵
    1. Lee SY,
    2. Obata Y,
    3. Yoshida M,
    4. Stockert E,
    5. Williamson B,
    6. Jungbluth AA,
    7. Chen YT,
    8. Old LJ,
    9. Scanlan MJ
    . Immunomic analysis of human sarcoma. Proc Natl Acad Sci U S A 2003;100:2651–2656.pmid:12601173
    OpenUrlAbstract/FREE Full Text
  37. 37.↵
      NCBI UniGene EST Profile Viewer entry for Hs.2324. URL: http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Hs.2324  
    1. 38.↵
      1. Hong HS,
      2. Chung WH,
      3. Hung SI,
      4. Chen MJ,
      5. Lee SH,
      6. Yang LC
      . Clinical association of anti-Golgi autoantibodies and their autoantigens. Scand J Immunol 2004;59:79–87.pmid:14723625
      OpenUrlPubMed
    2. 39.↵
      1. Fernandez-Madrid F,
      2. Tang N,
      3. Alansari H,
      4. Granda JL,
      5. Tait L,
      6. Amirikia KC,
      7. Moroianu M,
      8. Wang X,
      9. Karvonen RL
      . Autoantibodies to annexin XI-A and other autoantigens in the diagnosis of breast cancer. Cancer Res 2004;64:5089–5096.pmid:15289310
      OpenUrlAbstract/FREE Full Text
    3. 40.↵
      1. Caponi L,
      2. Giordano A,
      3. Bartoloni EB,
      4. Gerli R
      . Detection of anti-ribosome antibodies: a long story of lights and shadows. Clin Exp Rheumatol 2003;21:771–778.pmid:14740459
      OpenUrlPubMed
    PreviousNext
    Back to top
    Cancer Immunity Archive: 6 (1)
    January 2006
    Volume 6, Issue 1
    • Table of Contents

    Sign up for alerts

    View this article with LENS

    Open full page PDF
    Article Alerts
    Sign In to Email Alerts with your Email Address
    Email Article

    Thank you for sharing this Cancer Immunology Research article.

    NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

    Enter multiple addresses on separate lines or separate them with commas.
    Humoral immune responses to testis antigens in sera from patients with prostate cancer
    (Your Name) has forwarded a page to you from Cancer Immunology Research
    (Your Name) thought you would be interested in this article in Cancer Immunology Research.
    CAPTCHA
    This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
    Citation Tools
    Humoral immune responses to testis antigens in sera from patients with prostate cancer
    Luke H. Hoeppner, Jason A. Dubovsky, Edward J. Dunphy and Douglas G. McNeel
    Cancer Immun January 1 2006 (6) (1) 1;

    Citation Manager Formats

    • BibTeX
    • Bookends
    • EasyBib
    • EndNote (tagged)
    • EndNote 8 (xml)
    • Medlars
    • Mendeley
    • Papers
    • RefWorks Tagged
    • Ref Manager
    • RIS
    • Zotero
    Share
    Humoral immune responses to testis antigens in sera from patients with prostate cancer
    Luke H. Hoeppner, Jason A. Dubovsky, Edward J. Dunphy and Douglas G. McNeel
    Cancer Immun January 1 2006 (6) (1) 1;
    del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
    • Tweet Widget
    • Facebook Like
    • Google Plus One

    Jump to section

    • Article
      • Abstract
      • Introduction
      • Results
      • Discussion
      • Materials and methods
      • Acknowledgments
      • References
    • Figures & Data
    • Info & Metrics
    • PDF
    Advertisement

    Related Articles

    Cited By...

    More in this TOC Section

    • NY-ESO-1–specific immunological pressure and escape in a patient with metastatic melanoma
    • Hsp72 mediates stronger antigen-dependent non-classical MHC class Ib anti-tumor responses than hsc73 in Xenopus laevis
    • Human ovarian tumor ascites fluids rapidly and reversibly inhibit T cell receptor-induced NF-κB and NFAT signaling in tumor-associated T cells
    Show more Articles
    • Home
    • Alerts
    • Feedback
    • Privacy Policy
    Facebook   Twitter   LinkedIn   YouTube   RSS

    Articles

    • Online First
    • Current Issue
    • Past Issues
    • Cancer Immunology Essentials

    Info for

    • Authors
    • Subscribers
    • Advertisers
    • Librarians

    About Cancer Immunology Research

    • About the Journal
    • Editorial Board
    • Permissions
    • Submit a Manuscript
    AACR logo

    Copyright © 2021 by the American Association for Cancer Research.

    Cancer Immunology Research
    eISSN: 2326-6074
    ISSN: 2326-6066

    Advertisement