Introduction

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Mesothelial tissues include all those that line the cavities that are derived from the embryologic mesodermal coelomic cavity. Malignant mesothelioma (MM) usually develops in the pleura and occurs less commonly in other mesothelial tissues (1). The disease is associated with exposure to asbestos and is unresponsive to all conventional therapies, but there is some hope that in the future, MM may be effectively treated by immunologic approaches (2). This expectation arises from two types of evidence: first, clinical trials of various immunotherapeutic regimens in patients with MM have shown some capacity to ameliorate the disease (3). Second, the growth of transplantable syngeneic murine MM cell lines that induce a disease that is pathologically identical to the human disease (6) can be regulated by immunologic processes (7).

Many patients with MM respond immunologically to the presence of their tumor with no intervention. In our recent study of serologic responses by Western blot, we found that 28% of patients had serum antibodies of the immunoglobulin (Ig)G class in high titer (11). These findings are consistent with SEREX screens, which also suggest that many human tumors elicit multiple immune responses in the autologous host (12). SEREX is the serologic identification of antigens by recombinant expression cloning and can be readily applied to define tumor-associated antigens (TAA) at the molecular level.

There may be a number of reasons why TAA could be useful. First and most obvious, TAA are required for any targeted immunotherapeutic regimen. Second, antigens are required for vaccination of "at-risk" populations, and in MM these can be reasonably accurately defined because of the close correlation between exposure to asbestos and the development of the disease. Third, antigens might be useful for diagnosis or prognosis in that an antibody response might be expected to predate symptoms and clinical presentation. Serologic responses could be measured reasonably quickly and may be superior to more invasive assessment. There is no a priori reason why these sets of antigens should overlap. It seems likely that vaccination and immunotherapy will depend upon the generation of effector cells that include cytotoxic T lymphocytes (CTL). The recent cloning of TAA from other immunotherapy-sensitive cancers has resulted in an increased interest in the potential of active immunization as a strategy to control cancer (13). In particular, TAA have been cloned and characterized from melanoma (16), and antigen-specific CTL can be expanded in vitro using synthetic epitope peptides derived from such cloned sequences (21). Recent work has shown that appropriate presentation of whole antigen as well as peptide fragments can generate a tumoricidal CTL response (15). We have shown in our animal model of MM that transfected tumor antigens are constitutively presented to lymphocytes (22) and that tumor-specific CD4 cells greatly enhance the eradication of the tumor, confirming the importance of antigen-presenting cell presentation of tumor antigens to class II-restricted cells (23). Thus, there is now ample evidence that an antitumor immune response can lead to tumor regression, and the challenge for MM research is to define a useful set of target antigens.

In order to identify antigens associated with MM (MMAA), we applied the SEREX technique using sera from six patients with antibodies to MMAA identified in our previous study (11). This study clearly demonstrated that four of the six antisera were able to bind a homologous protein in both humans and mice. Because we ultimately want to test vaccination strategies in our mouse model of MM, we constructed our library from a cell line (AB1) that was derived after mice were injected intraperitoneally with crocidolite asbestos (6).

	Materials and Methods

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Sera and Patients

Blood was obtained by venipuncture from patients attending the respiratory clinic at the Queen Elizabeth II Medical Centre (QEII) in Perth. The type and stage of MM at diagnosis was identified by histologic examination of tumor samples and by radiology. The patients (13 men and one woman) were between 40 and 81 yr of age. Control subjects were selected from healthy lab volunteers between 29 and 58 yr of age. Blood was allowed to clot at room temperature and the clots retracted overnight at 4°C. Sera were clarified by centrifugation and stored in aliquots at 20°C. Parameters of systemic inflammation (erythrocyte sedimentation rate [ESR], platelet count, and peripheral blood white cell count [WCC]) were measured by the Westergren method (ESR), and platelet and WCC were measured by Coulter Gen.S autoanalyzer (Beckman Coulter, Inc., Fullerton, CA) in the Pathology Department at QEII. Tumor bulk was assessed by thoracic computed tomography scanning at the time the sera were taken and, for the purposes of this study, graded on a 0 to 5 scale as follows: 0 = no tumor mass; 1 = localized mass < 1 cm width; 2 = diffuse mass < 1 cm width; 3 = mass 1 to 2 cm width, regardless of extent; 4 = mass > 2 cm width, regardless of extent.

Cells

Various transformed cell lines were maintained in liquid culture by growing them at 37°C in a water-saturated atmosphere of 5% CO₂ in air. Cell lines derived from pleural effusions of patients with MM were adapted for growth in vitro (24). All other cell lines were sourced from the American Type Culture Collection (Manassas, VA). Cells were grown in RPMI 1640 containing 10% fetal calf serum supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine (all tissue culture media were supplied by Life Technologies, MD). Adherent cells were washed gently with phosphate-buffered saline and then removed by scraping with a sterile policeman.

Northern Blot Analysis

Total RNA was prepared from in vitro cultured cell lines and from various tissues of a mouse using the RNAzol protocol. The polyadenylated fraction was further purified by affinity adsorption to oligo dT magnetic beads (Dynabeads; Dynal, Oslo, Norway). RNA (5 µg per lane) was fractionated on a 1% agarose/ formaldehyde gel and transferred to a nylon membrane (Genescreen; DuPont). The random hexamer method was used to label probes representing the complementary DNAs (cDNAs) with [³²P]deoxyadenosine triphosphate. Probes were applied to the blot in 50% formamide hybridization solution at 42°C for 16 h as recommended by the manufacturer. Blots were washed in 0.2 × saline sodium citrate, 1% sodium dodecyl sulfate at 55°C, and analyzed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The same blot was serially hybridized with each probe; each probe was stripped by boiling the blot in water for 5 min before subsequent hybridization. A probe made from the cDNA encoding the ribosomal S26 protein was used as an internal loading control.

Construction of  Expression Library

cDNA was prepared from the murine MM cell line AB1 using XhoI-tagged oligo dT as a primer. The derivation and characterization of this cell line has been described in detail elsewhere (6). cDNA was blunted, capped with EcoRI adapters, and ligated into the zap vector (Stratagene, La Jolla, CA). The library, with a complexity in excess of 1 × 10⁶, was amplified, and the phage stored at 4°C at a concentration of 1 × 10¹⁰ PFU/ml.

Screening of the Library with Serum

Six sera that reacted positively in Western blot to MM cell lysates were mixed at a dilution of 1:100. To remove any background reactivity to bacterial proteins, antibodies from the serum pool were absorbed onto an Escherichia coli lysate by admixture followed by centrifugation. The process was repeated three times. Plaques 3 × 10⁶ were screened using the serum pool and identified using an alkaline phosphatase conjugated antihuman IgG (Promega, Madison, WI) and the picoBLUE immunoscreening kit (Stratagene). Positive clones from the primary screen were picked and replated over several rounds until they were monoclonal. In vivo excision of the purified plaques as the pBluescript phagemid was carried out using the ExAssist helper phage as described by the manufacturer (Stratagene). Inserts were sequenced using dideoxy chain termination.

Freckle Assay

Clonal phage preparations were titrated and applied to a bacterial lawn using a plate replicator to achieve a plaque density of around 100 cm². Individual filter lifts containing plaques from each of the clones of interest were then tested with each of the sera and developed as above. A reaction was scored on a scale from negative to +++ within each filter.

	Results

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Identification of Antigens Associated with MM

A  expression library constructed from the MM cell line AB1 with a complexity in excess of one million was plated at a density of 3,000 plaques cm². Plaques 2 × 10⁶ were tested for reactivity with a serum pool constructed from six patients with MM, each diluted 1:100. A total of 93 primaries was picked, of which 12 retested positive in third round screening at which stage they were all clonal. Purified plaques were excised as the pBluescript plasmid and subject to sequence analysis. Eight different cDNAs were identified from the sequence information (Table 1). Topoisomerase II (TOPII) was picked four times from different primary plaques; three of the four picks were different recombinants. Siah binding protein (SBP) was picked twice as an independent recombinant, indicating that the library was not biased. Two novel transcripts were identified and named Jemm-1 and Jemm-2. Each plasmid contained an insert with an open reading frame that was translatable as an in-frame fusion with the N terminus of the -galactosidase protein encoded by the vector. Jemm-1 was matched to a single expressed sequence tag (EST) from a library created from mouse myotubes; Jemm-2 was not represented in any EST database even as a weak homologue and was therefore deposited with Genbank (Table 1).

View larger version (25K): [in this window] [in a new window]   View larger version (29K): [in this window] [in a new window]   Figure 1.   Northern blot analysis to determine the pattern of expression of various genes (as indicated) in normal tissue and in cell lines characteristic of MM. (A, B, and D) Blots were prepared from polyadenylated RNA (5 µg/lane). (C ) Blot was prepared from total RNA (20 µg/lane). Because of the low expression of Jemm-2, B was digitally enhanced to remove washing artifacts.

Patterns of Expression

The tissue specificity of expression of each of the antigens was investigated by Northern blot analysis. In some cases where the level of expression was low, blots were prepared from polyadenylated RNA. The antigens segregated equally into two categories, those that were overexpressed in one or more cell lines characteristic of MM (Figures 1A-1C) and those that were not (Figure 1D). The two novel transcripts plus messenger RNA (mRNA) for U2AF(65) and mIre1 were overexpressed in some of the MM cell lines. Ire1, Jemm-1, and Jemm-2 show a limited tissue distribution, but only Jemm-1 has low expression in the thymus. The four genes that were found not to be overexpressed in any of the MM cell lines exhibited variable patterns of expression in normal mouse tissue. Of particular note, pendulin was found to be highly expressed in the testis, and tissue-specific transcripts of the ZFM-1 gene were found in the lung and testis.

Serologic Recognition of the Antigens

We then investigated whether the occurrence of antibodies to each of the antigens was associated with individual cases of MM. Purified phage isolates were allowed to form plaques that were induced with isopropyl B-D-thiogalactoside (IPTG), and the recombinant proteins were transferred to nitrocellulose membranes. Replicate membranes were probed with serum (1:50 dilution) and processed as described in MATERIALS AND METHODS. Plaques were scored positive by visual inspection and in relation to each of the other plaques on the membrane. Thirteen of 14 (93%) patients with MM had antibodies to TOPII, whereas only six of 16 (37%) healthy laboratory volunteers scored positive. Antibodies to U2AF(65) were found in two patients, but antibodies to the other antigens were found in only a single patient, and none in the control group scored positive. Of the original six sera used for probing the library, all were positive for TOPII, and only one did not recognize an additional antigen. Interestingly, the complexity of the immune response correlated with patient survival (Table 2). The mean survival time from diagnosis was longest in those individuals manifesting more than one serum reactivity. There was no correlation between the number of serum reactivities and tumor bulk. Whereas tumor bulk is one way of measuring disease, it is known that not all patients with the same amount of tumor have identical clinical features, with systemic features often being associated with evidence of host inflammatory responses such as elevated ESR, platelet count, and peripheral blood WCC. We therefore determined whether the patients' serologic reactivities paralleled the presence of these inflammatory parameters. Patients with two or more reactivities had a higher ESR than those with fewer reactivities, perhaps reflecting a more active host immune response to the presence of tumor.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Tumor-Associated Antigens

TAA are important for a number of reasons. The most widely cited is that they offer potential targets for the immune system. These targets may be useful for directing immunotherapy in patients already diagnosed with disease (type I antigens), or they may be useful for generating immune responses in healthy individuals who are at risk of developing the disease (type II antigens). An equally valid reason to identify TAA is that an understanding of the immune response and, in particular, the serologic response to some TAA could have wide implications for the diagnosis and prognosis of cancer (type III antigens). Although there is no a priori reason to assume that TAA that are useful for immunotherapy (type I antigens) will be good candidates for vaccination programs (type II antigens), they may well fall into overlapping groups. This is because the mode of the immune response that is sought for these TAA is of the T helper (Th)1/CTL type that is characteristic of viral infections. In contrast, TAA that are useful in diagnosis and prognosis (type III antigens) because they generate autoantibodies are likely to be characterized at the T-cell level by Th2 responses that are normal sequelae to bacterial infections. Such antigens might not therefore be expected to overlap with type I and type II antigens.

Some of the genes we have identified in this study are likely required by and are therefore expressed by all cells, although the Northern blots demonstrate that their level of expression can vary in different tissues. Other genes identified in this study are differentially expressed. The expression of Jemm-1, for example, is limited to a pattern consistent with the distribution of mesothelial tissue. Exposing an antibody reactivity to a common antigen may also be informative about MM. Autoantibodies to commonly expressed antigens are not unusual in autoimmune (AI) disease where a particular organ is specifically targeted. For example, antibodies to mitochondrial antigens are common in AI liver disease, and antibodies to amino acyl transfer RNA transferases are a feature of cryptogenic fibrosing alveolitis. We think that the interesting and unusual pattern of reactivity in MM is a starting point for further study.

Autoantibodies are a feature of a variety of AI diseases, but perhaps, surprisingly, the antibodies can appear long before the manifestation of any pathology. In diabetes, for example, antibodies against insulin and other islet cell antigens become detectable months to years before clinically detectable disease (25). It seems reasonable to hypothesize that patients with malignant changes or precancerous lesions respond immunologically to these changes without necessarily showing any clinical features of their disease. The best evidence that this is indeed the case has come from studies of the serologic response to p53 (26). Thus, TAA derived using serum as a probe may be useful prognostically if it can be demonstrated that a majority of patients make responses to an overlapping set of antigens. We probed a mouse library because we anticipated a greater utility for mouse homologs of MMAA as vaccines in our murine model of MM. This strategy may in itself have lowered the sensitivity of the analysis in that we might expect to identify only antigens with conserved B-cell epitopes. However, we have already shown that serologic responses are common in patients with MM and that many patients' sera identify homologous proteins in the mouse (11); so we expect that most of the reactivities we have found are relevant to both mice and humans.

Serologic Response to the Presence of Tumor

Searching for TAA using an antibody response to probe for the antigens may be the most powerful way to identify antigens of the third type. This is simply because these antigens are already defined as targets for a serologic response. The major factors in determining their usefulness will be defined by characterizing the frequency of the response to each of these TAA. Antibodies have been described that react with many different normal cellular proteins; some of these proteins are known to be overexpressed in tumors. Those that have excited most interest identify antigens of oncofetal origin or with restricted tissue distribution because of the expectation that they will have properties consistent with type I and type II antigens. Historically, there has been some expectation that mutated proteins involved in tumorigenesis would provide a unique target to activate the immune system. At least for some oncogene products, this expectation was ill founded, although there are isolated reports of novel T-cell epitopes associated with activating mutations of some genes (27). In general, it seems that the B-cell response to TAA is focused on regions located outside the mutational hot spots (28), indicating that a sustained response may depend on overexpression of a protein rather than de novo expression of a neo epitope. We have no way to find out the level of expression of any of the genes we have found in the primary tumor of the patients that provided the sera. We can hypothesize that those tumors overexpressed the genes to which the patients made an antibody response, and this is consistent with the finding that the mouse tumor cell lines are heterogeneous in their expression of each of the antigens. To investigate the possibility of unique specificities, we plan to screen patients against their own primary tumor.

MMAA Identified in This Study

U2AF(65). This is the 65-kD subunit of a functional heterodimer expressed ubiquitously. This pattern of expression is to be expected because U2AF plays a central role in the normal cellular pre-mRNA splicing process (29). This protein has been identified as an antigen in a patient with hepatocellular carcinoma, and in the same way described here, antibodies from serum were used to isolate the cDNA (30). WT1 is considered a transcription factor and is associated with the development of Wilm's tumor in the kidney. WT1 is also expressed in MM (31). Interestingly, WT1 also interacts with U2AF(65) and associates with the splicing machinery (32). WT1 binds to and stabilizes p53; it modulates the transactivational properties of p53 and can inhibit its ability to induce apoptosis (33).

SBP1. The Drosophila seven in absentia (sina) gene is required for R7 photoreceptor cell formation during Drosophila eye development, where it functions within the Ras/ Raf pathway, targeting proteins for degradation by ubiquitination. Proteins in this pathway are highly conserved, and the human homologs of SINA are called SIAH. SIAH proteins are expressed in many normal and neoplastic tissues. Their substantial evolutionary conservation, their role in specifying cell fate, and their activation in apoptotic cells suggest they have important roles in vertebrate development. SIAH proteins may be inducible by p53 and therefore involved in p53-dependent cell-cycle arrest (34). The clone that we have found, SBP1, was originally isolated using a yeast, two-hybrid system with SIAH as bait (see notes in Genbank under accession number U51586).

TOPII. Type II DNA topoisomerases reformat DNA topologically. The enzymes coordinate relaxation of DNA supercoils generated in transcription and replication, and they play an essential role in the condensation of chromosomes and their segregation during mitosis. In mammals, this activity is derived from at least two isoforms, termed alpha and beta. The alpha isoform is involved in chromosome condensation and segregation, whereas the role of the beta isoform is not yet clear (35). It is interesting that p53 can be coimmunoprecipitated with TOPII, and the interaction appears not to require a DNA intermediary. Furthermore, TOPII proteins have been found to be overexpressed in the majority of hepatocellular carcinomas, including several in which presumed wild-type p53 was detected by immunohistochemistry (36). We are currently investigating the status of TOPII expression in human MM cell lines and primary tumors.

ZFM1. This protein has been identified independently as both a transcriptional repressor and as the splicing factor SF1 (37). SF1 binds specifically to branchpoint sequences during RNA splicing and interacts with U2AF(65). The SF1-U2AF(65) interaction promotes cooperative binding to a branchpoint sequence-polypyrimidine tract- containing RNA (38). As noted previously, these activities are associated with the binding of WT1 and may be regulated by p53. In contrast to our study's Northern blot analysis, showing ZFM1 as widely expressed, previous studies have suggested that its expression is limited to spleen macrophages, although a low basal expression was noted in the majority of other tissues. Alternately spliced transcripts as shown in this article were also described (39). Differential display has linked the p53 signaling pathway with expression of ZFM1 and SINA (40).

mIre1. Nascent proteins often require the help of other factors to ensure that they fold correctly. A number of proteins have this task, including the heat shock proteins, chaperones such as BiP, and various isomerases including protein disulfide isomerase. The genes encoding these proteins are coordinately regulated by transcriptional activation. In yeast, this pathway operates through the ability of a transmembrane signaling protein (Ire1p) to recognize misfolded proteins in the lumen of the endoplasmic reticulum and transmit a signal, which is interpreted in the cell nucleus, by transcription factors. The cloning of a murine homolog of yeast Ire1 and the overexpression of a dominant negative form indicate that it is the proximal sensor of the endoplasmic reticulum stress-response in mammalian cells (41).

Pendulin. Pendulin, in association with other proteins, mediates the transfer to the nucleus of proteins containing the nine amino-acid nuclear localization signal (KKKKRKREK), such as the transcription factor lymphoid enhancer factor 1 (42). It is highly expressed in the testis, but we see no evidence of overexpression in any of the murine MM cell lines.

Jemm-1. This novel gene appears as an EST in Genbank (Table 1). The sequence was derived from mouse myotubes. The expression of this gene is limited to lung, kidney, heart, and ovary, a pattern consistent with the distribution of mesothelial tissue. Jemm-1 is also expressed in MM cell lines (Figure 1A). Translation of Jemm-1 reveals a weak homology to a previously identified MMAA (OVCAR-3) also expressed on ovarian cancers (43).

Jemm-2. The DNA sequence of this novel gene did not appear in any of the publicly available databases but is now lodged with Genbank. Translation and searching of the protein yields no matches in any frame in any database, and therefore there are no clues to its function. We find that the mRNA for Jemm-2 is only weakly expressed in spleen and thymus, and this may account for its nonappearance in the databases. Interestingly, the gene appears to be overexpressed in at least one MM cell line (Figure 1B).

Serologic Responses in MM

It is clear that none of the antigens detected in this study are uniquely expressed in MM. This finding accords with the findings of other investigators (44) and supports the notion that the immunogenicity of particular tumors is a consequence of overexpression of a normal self-protein rather than the novel expression of TAA. But beyond this, we conclude that the spectrum of overexpressed proteins is different in the cancers of different individuals. There was no obvious correlation between the age and sex of the patients and their immune response to their tumor. The relevance of the finding that increasing complexity of the serologic response to tumor is associated with survival is difficult to assess in this study because of the limited availability of serially obtained sera. On the one hand, it could suggest that an immune response is a good prognostic indicator; alternately, it may be that those patients who survive longer have more time to mount an immune response that is epiphenomenal to the pathogenesis. We consider this second possibility to be less likely because when serial samples are examined, we have only rarely noted a change in the pattern of reactivity in Western blot analysis, and we have seen loss of reactivity as commonly as gain of reactivity.

The stage of disease was not different in these patients. As a more accurate guide, we therefore assessed two clinical aspects and related them to antibody reactivity. First, we evaluated tumor bulk, and it is apparent that the broader pattern of reactivity is not a reflection of tumor bulk. Second, we evaluated systemic/inflammatory parameters, often considered to be a reflection of aggressive tumor. Interestingly, patients with two or more reactivities had a higher ESR than those with less reactivities, and as this did not correlate with tumor bulk itself, it suggests that the broader range of reactivities reflects a more active host response to the presence of tumor. Future studies will clarify these issues.

We found antibodies to TOPII in 37% of the control group. In general, these reactions were only weakly positive in the subjective analysis of the data (scoring either + or ++). In contrast, some of the patients with MM were strongly positive (scoring either ++ or +++). Even though the filters were read blindly, it was not possible to obtain meaningful scores because each filter was characterized by a different background that could only be judged against the other plaques on the same filter. An accurate determination of the magnitude of the serologic response to TOPII awaits the realization of a quantitative assay such as enzyme-linked immunosorbent assay, and we are engaged in this development.

We need to address the question, how do the MMAA identified here relate to our previous Western blot analysis (11)? At least superficially, we appear to be looking at the same proteins identified by the same set of patients. In Western blot analyses, six of the eight antigenic complexes that were studied were expressed at least partially in the nucleus. The cloning experiments have identified eight antigens, six of which would be expected to localize to cell nuclei. The Western blot analysis identified two antigen complexes with some degree of mesothelial tissue-specific expression. Using SEREX and Northern blot analysis, three of the eight antigens were found to be differentially expressed with overexpression in at least one MM cell line. Western blot analysis of bacterially expressed gene products showed that serum 15 and serum 27 (described in Reference 11) recognize SBP and U2AF, respectively; the other sera failed to react in this analysis (data not shown) presumably because the epitopes they detected were conformational and were denatured in the procedure (data not shown). Thus, the antigens detected in the original Western blot analysis of these sera likely constitute part of an overlapping set of antigens, some of which are not detected in denatured form.

In conclusion, we have characterized the serologic reactivity of patients with MM to proteins expressed by MM cells, identifying both public and private specificities. These reactivities might ultimately be useful in the diagnosis of MM. The next phase of our serologic analysis will be to determine the frequency of immune responses to the MMAA in patients with other cancers and in individuals exposed to asbestos who have a high risk of the development of MM. We will also determine the titer and class of antibodies to TOPII to find out if these correlate more closely with the disease. Some of the proteins identified by these sera have restricted tissue distribution and may therefore be useful in immunotherapy or for vaccination of "at-risk" individuals. We therefore also plan to assess the level of expression of the MMAA we have described in primary MM and in tumors of different origin.