Identification of a meiosis-specific protein as a member of the class of cancerytestis antigens

Little is known about the function of human cancerytestis antigens (CTAs), such as MAGE, BAGE, GAGE, HOM-MEL-40, and NY-ESO-1, the expression of which is restricted to human malignancies and testis. When screening a cDNA expression library enriched for testis-specific representative long transcripts for reactivity with high-titered IgG antibodies from the serum of a patient with renal cell carcinoma, one repeatedly detected antigen, designated HOMTES-14, turned out to be encoded by the synaptonemal complex protein 1 (SCP-1) gene. SCP-1 is known to be selectively expressed during the meiotic prophase of spermatocytes and is involved in the pairing of homologous chromosomes, an essential step for the generation of haploid cells in meiosis I. Investigation of a broad spectrum of normal and malignant tissues revealed expression of SCP-1 transcripts and antigen selectively in a variety of neoplastic tissues and tumor cell lines. Immunofluorescence microscopy analysis with specific antiserum showed a cell cycle phaseindependent nuclear expression of SCP-1 protein in cancer cells. SCP-1 differs from other members of the class of CTA by its localization on chromosome 1 and its frequent expression in malignant gliomas, breast, renal cell, and ovarian cancer. The aberrant expression of SCP-1 in tumors might contribute to their genomic instability and suggests that the functional role of other CTA might also relate to meiosis. The identification of genes that are selectively expressed in cancer and code for proteins inducing a specific immune response in the tumor-bearing host is the prerequisite for specific immunotherapeutic approaches to cancer. By using cytotoxic T lymphocyteand antibody-based approaches, several human tumor antigens have been defined that might be suitable targets for specific cancer vaccination (1, 2). The difficulties in establishing cytotoxic T lymphocyte clones with specificity to human neoplasms are the major reason why— with the exception of RAGE (renal cell cancer-associated antigen) (3)—all T cell-defined human tumor antigens have been originally described in malignant melanoma. Immunotherapeutic approaches to human neoplasms other than melanoma are restricted to the nonmelanoma cases that express the melanoma-defined antigens. To overcome this limitation, we recently established SEREX (serological analysis of antigens by recombinant expression cloning) that allows for the unbiased search of tumor antigens that elicit IgG antibody responses in the autologous tumor patients (4). With this approach, we have identified multiple tumor antigens in different human neoplasms. Among others, we detected MAGE-1 (melanoma-associated antigen) and MAGE4a transcripts, demonstrating that at least some of the serologically identified antigens are also targets for cytotoxic T cells. The analysis of the expression pattern of SEREX antigens revealed that a group of tumor antigens, such as HOM-MEL-40, NY-ESO-1, and several other (unpublished results) antigens, are selectively expressed in a variety of human neoplasms but not in normal tissues except for testis. This characteristic expression spectrum has also been described for the T celldefined MAGE, BAGE, and GAGE gene products and has led to the designation of the term cancerytestis antigens (CTAs; ref. 5). Besides differentiation antigens such as Melan AyMART-1, tyrosinase, gp100yPmel17, and gp75 (6–11), CTA respresent a second major class of molecularly defined human tumor antigens. Although the function of many differentiation antigens is known, the function of all the CTAs has resisted scrutiny. The selective expression of CTAs in a broad spectrum of neoplasms makes them ideal candidates for specific cancer immunotherapy. However, because the known CTA are expressed in only a small spectrum of human cancers and in only a fraction of cases of a given tumor type, the identification of additional tumor antigens is badly needed. The fact that all CTAs are also expressed at high levels in testis prompted us to screen a testis expression library enriched for specific transcripts instead of a cDNA library derived from a tumor. This approach has led to the identification of several more CTAs. One of these antigens, HOM-TES-14, is encoded by synaptonemal complex protein 1 (SCP-1), a gene specifically expressed in the meiotic prophase of spermatocytes. The SCP-1 protein is involved in the meiotic chromosome synapsis of sperm cells. MATERIALS AND METHODS Sera, Tissues, and Cell Lines. The study had been approved of by the local ethical review board (‘‘Ethikkommission der Ärztekammer des Saarlandes’’). Recombinant DNA work was done with the official permission and according to the rules of the state government of Saarland. Sera and tumor tissues were obtained during routine diagnostic or therapeutic procedures and were stored at 280°C until use. Normal tissues were collected from autopsies of tumor-free patients. Construction of the Subtractive cDNA Expression Library. For the construction of the subtractive cDNA expression library, a modification of the recently described suppression subtractive hybridization (SSH) technique (12) was used. In brief, a pool of testis-specific cDNA fragments were amplified by the SSH technique. These cDNA fragments were used to capture their long counterparts from a cDNA phagemid library. Finally, cDNA inserts excised from captured phagemids were cloned into l phage vectors and used for expression The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y955211-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: CTA, cancerytestis antigen; MAGE, melanomaassociated antigen; SCP-1, synaptonemal complex protein 1; SEREX, serological analysis of antigens by recombinant expression cloning; PBMC, peripheral blood mononuclear cell. †O.T. and U.S. contributed equally to this work. ‡To whom reprint requests should be addressed. e-mail: UgurSahin@ aol.com.