A putative tumor suppressor locus on the short arm of human chromosome 9 has been localized to a region of less than 40 kilobases by means of homozygous deletions in melanoma cell lines. This region contained a gene, Multiple Tumor Suppressor 1 (MTS1), that encodes a previously identified inhibitor (p16) of cyclin-dependent kinase 4. MTS1 was homozygously deleted at high frequency in cell lines derived from tumors of lung, breast, brain, bone, skin, bladder, kidney, ovary, and lymphocyte. Melanoma cell lines that carried at least one copy of MTS1 frequently carried nonsense, missense, or frameshift mutations in the gene. These findings suggest that MTS1 mutations are involved in tumor formation in a wide range of tissues.

This study demonstrates that endogenously produced interferon ␥ (IFN-␥) forms the basis of a tumor surveillance system that controls development of both chemically induced and spontaneously arising tumors in mice.

Serological analysis of recombinant cDNA expression libraries (SEREX) using tumor mRNA and autologous patient serum provides a powerful approach to identify immunogenic tumor antigens. We have applied this methodology to a case of esophageal squamous cell carcinoma and identified several candidate tumor targets. One of these, NY-ESO-1, showed restricted mRNA expression in normal tissues, with high-level mRNA expression found only in testis and ovary tissues.

Evidence is growing for both humoral and cellular immune recognition of human tumor antigens. Antibodies with specificity for antigens initially recognized by cytotoxic T lymphocytes (CTLs), e.g., MAGE and tyrosinase, have been detected in melanoma patient sera, and CTLs with specificity for NY-ESO-1, a cancer-testis (CT) antigen initially identified by autologous antibody, have recently been identified. To establish a screening system for the humoral response to autoimmunogenic tumor antigens, an enzyme-linked immunosorbent assay (ELISA) was developed using recombinant NY-ESO-1, MAGE-1, MAGE-3, SSX2, Melan-A, and tyrosinase proteins. A survey of sera from 234 cancer patients showed antibodies to NY-ESO-1 in 19 patients, to MAGE-1 in 3, to MAGE-3 in 2, and to SSX2 in 1 patient. No reactivity to these antigens was found in sera from 70 normal individuals. The frequency of NY-ESO-1 antibody was 9.4% in melanoma patients and 12.5% in ovarian cancer patients. Comparison of tumor NY-ESO-1 phenotype and NY-ESO-1 antibody response in 62 stage IV melanoma patients showed that all patients with NY-ESO-1+ antibody had NY-ESO-1+ tumors, and no patients with NY-ESO-1− tumors had NY-ESO-1 antibody. As the proportion of melanomas expressing NY-ESO-1 is 20–40% and only patients with NY-ESO-1+ tumors have antibody, this would suggest that a high percentage of patients with NY-ESO-1+ tumors develop an antibody response to NY-ESO-1.

A growing number of human tumor antigens have been described that can be recognized by cytotoxic T lymphocytes (CTLs) in a major histocompatibility complex (MHC) class I–restricted fashion. Serological screening of cDNA expression libraries, SEREX, has recently been shown to provide another route for defining immunogenic human tumor antigens. The detection of antibody responses against known CTL-defined tumor antigens, e.g., MAGE-1 and tyrosinase, raised the question whether antibody and CTL responses against a defined tumor antigen can occur simultaneously in a single patient. In this paper, we report on a melanoma patient with a high-titer antibody response against the “cancer–testis” antigen NY-ESO-1. Concurrently, a strong MHC class I–restricted CTL reactivity against the autologous NY-ESO-1–positive tumor cell line was found. A stable CTL line (NW38-IVS-1) was established from this patient that reacted with autologous melanoma cells and with allogeneic human histocompatibility leukocyte antigen (HLA)-A2−, NY-ESO-1–positive, but not NY-ESO-1–negative, melanoma cells. Screening of NY-ESO-1 transfectants with NW38-IVS-1 revealed NY-ESO-1 as the relevant CTL target presented by HLA-A2. Computer calculation identified 26 peptides with HLA-A2–binding motifs encoded by NY-ESO-1. Of these, three peptides were efficiently recognized by NW38-IVS-1. Thus, we show that antigen-specific humoral and cellular immune responses against human tumor antigens may occur simultaneously. In addition, our analysis provides a general strategy for identifying the CTL-recognizing peptides of tumor antigens initially defined by autologous antibody.

A nalysis of spontaneous immune responses against cancer in humans has led to the identification of a large number of tumor antigens (1). The majority of these antigens can be classified into one of the following categories according to their expression pattern, function, or origin: cancer-testis (CT) antigens, e.g., MAGE (2, 3) and NY-ESO-1 (4), which are aberrantly expressed in tumor cells but that, with the exception of germ cells, are silent in normal cells; differentiation antigens of the melanocyte lineage, e.g., Melan A͞MART-1 (5, 6), tyrosinase (7), and gp100 (8, 9); mutational antigens, e.g., MUM-1 (10), p53 (11, 12), and CDK4 (13); overexpressed ''self'' antigens, e.g., HER2͞neu (14) and p53 (12); and viral antigens, e.g., HPV (15) and EBV (16). Spontaneous immune responses elicited by these antigens are either predominantly cellular, e.g., tyrosinase (17, 18) and Melan A͞MART-1 (9, 19), or are associated with a strong humoral immune component, e.g., NY-ESO-1 (20) and p53 (12).NY-ESO-1 is a highly immunogenic CT antigen, inducing simultaneous cellular and humoral immune responses in a high percentage of patients with advanced NY-ESO-1-expressing tumors (20,21). Detectable NY-ESO-1 serum antibody depends on the presence of NY-ESO-1-expressing tumor, and antibody titers correlate with the clinical development of disease (20,22). NY-ESO-1-specific CD8ϩ T-cell responses were detected in more than 90% of NY-ESO-1 antibody-positive patients, whereas NY-ESO-1 antibody-negative patients showed no detectable NY-ESO-1-specific T-cell reactivity (23).The present study was initiated to evaluate the effects of active immunization with NY-ESO-1 peptides in NY-ESO-1 antibodynegative and -positive patients. Three naturally processed NY-ESO-1 peptides presented by HLA-A2 were used for intradermal immunization, first alone and then in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF) as a systemic adjuvant. The following parameters were monitored in this trial: (i) peptide-specific CD8ϩ T-cell responses; (ii) delayed-type hypersensitivity (DTH) reactivity; (iii) NY-ESO-1-specific antibody responses; and (iv) disease status. MethodsImmunization Protocol. Twelve HLA-A2ϩ patients with progressing NY-ESO-1-expressing metastatic tumors of different types and meeting predefined entry criteria were selected for immunization in the LUD97-008 protocol sponsored by the Ludwig Institute for Cancer Research. Immunizations were performed with three HLA-A2-binding NY-ESO-1 peptides derived from NY-ESO-1 and initially identified by the T-cell line NW38-IVS-1 (21). The NY-ESO-1 peptide sequences were: p157-167 (SLLM-WITQCFL), p157-165 (SLLMWITQC), and p155-163 (QLSLLMWIT). The HLA-A2-presented influenza matrix peptide p58-66 (GILGFVFTL) was used as a positive control for immune responses in vitro and in vivo. Peptides (Ͼ90% purity) were manufactured according to good manufactorial practice guidelines (Multiple Peptide Systems, San Diego) and solubilized in 100% DMSO. Intradermal injection of the 100% DMSO͞pepti...

M ajor progress in the identification and characterization of human tumor antigens has occurred over the past decade (1). The development of approaches to analyzing humoral (2) and cellular (3) immune reactivity to cancer in the context of the autologous host has led to the molecular characterization of tumor antigens recognized by CD8 ϩ T cells (4) and antibody (5). As a consequence of these advances, it is now possible to classify human tumor antigens, and the majority of antigens defined to date fall into one or more of the following categories: (i) differentiation antigens, e.g., tyrosinase (6), Melan-A͞MART-1 (7, 8), and gp100 (9); (ii) mutational antigens, e.g., CDK4 (10), ␤-catenin (11), caspase-8 (12), and p53 (13); (iii) amplification antigens, e.g., HER2͞neu (14) and p53 (15) (29), and CT10͞ MAGE-C2 (30). Six of these CT systems are known to be coded for by genes on the X chromosome. The only CT antigen with a known function is SCP-1, a synaptonemal complex protein involved in chromosome reduction in meiosis (26).NY-ESO-1, the focus of the present study, was identified during a SEREX analysis of an esophageal cancer (20). The gene for NY-ESO-1 maps to Xq28 (31) and codes for an 18-kDa protein (20). NY-ESO-1 mRNA expression is found in 20-30% of melanomas, lung, breast, ovarian, and bladder cancers, and other tumor types but, like other CT antigens, rarely in colon cancer or renal cancer (20). In a survey of sera from normal individuals and cancer patients, antibodies to NY-ESO-1 were found in 40 -50% of patients with advanced NY-ESO-1-expressing tumors (32). One patient with high-titered NY-ESO-1 antibodies was found to have HLA-A2-restricted cytotoxic T lymphocytes against autologous NY-ESO-1-expressing melanoma cells, and three HLA-A2-restricted NY-ESO-1 peptides were identified as the target epitopes recognized by these cytotoxic T lymphocytes (33).In the present study, we have analyzed CD8 ϩ T cell responses to NY-ESO-1 by enzyme-linked immunospot (ELISPOT), cytotoxicity, and tetramer assays and the humoral immune responses by ELISA and Western blots. Our findings show that NY-ESO-1 elicits a strong, integrated humoral and cellular immune response in a high proportion of patients with NY-ESO-1-expressing tumors. Materials and MethodsTumor Typing for NY-ESO-1 mRNA. Expression of NY-ESO-1 mRNA in tumor specimens was assessed by reverse transcription-PCR, using previously described primers (20).Assays for NY-ESO-1 Antibody. NY-ESO-1 serum antibodies were assayed by ELISA and Western blots, using NY-ESO-1 recombinant protein purified from Escherichia coli (32).Abbreviations: ELISPOT, enzyme-linked immunospot; PBL, peripheral blood lymphocyte; SEREX, serological screening of cDNA expression libraries; CT, cancer-testis. † E.J. and Y.N.

Cancer/testis (CT) antigens are expressed in a range of malignant neoplasms, but with the exception of testis, not in normal tissues. 1,2 CT antigens elicit cellular 3 as well as humoral 4 -6 immune responses. Ten genes or gene families have been identified that code for CT antigens. 7 The first members of this class of tumor antigens, MAGE, BAGE and GAGE, were identified as targets for CD8 T cells in a patient with melanoma. 8 -10 Other CT antigens have been recognized by the cloning technique called SEREX, involving serological screening of cDNA expression libraries of tumors with patient sera as the source of antibody 11 and by representational-difference analysis. 12,13 NY-ESO-1 is a classic CT antigen discovered during a SEREX analysis of an esophageal cancer. 14 NY-ESO-1 is a 22 kD hydrophobic protein 14 coded for by a gene in the Xq28 region. 15 In normal tissues, expression of NY-ESO-1 mRNA as detected by RT-PCR is predominantly restricted to the testis, whereas in cancer NY-ESO-1 is found in a variable proportion of a wide range of different malignancies, including melanoma, cancers of the breast, ovary and lung. 14 The strong immunogenicity of NY-ESO-1 is another distinguishing feature of the antigen; 40 -50% of patients with advanced NY-ESO-1 mRNA positive tumors have humoral 6,16,17 or CD8 T cell 18 response to NY-ESO-1.Current knowledge about CT antigen expression in normal and malignant tissues is mainly based on RT-PCR analysis. Mouse monoclonal antibodies (MAbs) to MAGE gene products have been generated, 19 -23 and these have been used to analyze the pattern of MAGE expression in tissues. 24,25 Mouse MAbs recognizing NY-ESO-1 antigen have been generated against NY-ESO-1 recombinant protein. 6 In the present study, these NY-ESO-1 MAbs have been used to establish the expression pattern of NY-ESO-1 in normal and malignant tissues. MATERIAL AND METHODS AntibodiesTwo NY-ESO-1 monoclonal antibodies were described previously. 6 Clone E978 (IgG1) was generated against a 23 kD NY-ESO-1 recombinant protein and clone ES121 (IgG1) was generated against a shorter, 14 kD NY-ESO-1 recombinant protein.Initial analysis with testis and selected tumor tissues indicated a similar immunohistochemical staining pattern with these two MAbs. Because ES121 showed both the best and most consistent results in immunohistochemical staining, it was chosen as the reagent for our study. TissuesFrozen and formalin-fixed paraffin-embedded tissues were provided by the Departments of Pathology of Memorial Sloan-Kettering Cancer Center and New York Hospital Cornell University Medical School. Frozen and 10% neutral buffered formalin-fixed paraffin-embedded tissues were used. The frozen specimens were embedded in O.C.T. (Tissue Tek, Torrance, CA) and snap-frozen in pre-cooled (Ϫ70°C) isopentane. Five m cuts were performed from frozen or paraffin blocks and applied to histology slides for immunohistochemistry (Superfrost Plus, Fisher Scientific, Pittsburgh, PA). Frozen sections were fixed in cold acetone for 10 min and air-dried befor...