Purpose: Antibody-based cancer therapies have emerged as the most promising therapeutics in oncology. The purpose of this study was to discover novel targets for therapeutic antibodies in solid cancer. Experimental Design:We combined data mining and wet-bench experiments to identify strictly gastrocyte lineage^specific cell surface molecules and to validate them as therapeutic antibody targets. Results: We identified isoform 2 of the tight junction molecule claudin-18 (CLDN18.2) as a highly selective cell lineage marker. Its expression in normal tissues is strictly confined to differentiated epithelial cells of the gastric mucosa, but it is absent from the gastric stem cell zone. CLDN18.2 is retained on malignant transformation and is expressed in a significant proportion of primary gastric cancers and the metastases thereof. In addition to its orthotopic expression, we found frequent ectopic activation of CLDN18.2 in pancreatic, esophageal, ovarian, and lung tumors, correlating with distinct histologic subtypes. The activation of CLDN18.2 depends on the binding of the transcription factor cyclic AMP^responsive element binding protein to its unmethylated consensus site. Most importantly, we were able to raise monoclonal antibodies that bind to CLDN18.2 but not to its lung-specific splice variant and recognize the antigen on the surface of cancer cells. Conclusions: Its highly restricted expression pattern in normal tissues, its frequent ectopic activation in a diversity of human cancers, and the ability to specifically target this molecule at the cell surface of tumor cells qualify CLDN18.2 as a novel, highly attractive pan-cancer target for the antibody therapy of epithelial tumors.Gastroesophageal and pancreatic cancers are among the malignancies with the highest unmet medical needs (1). Gastric cancer is the second leading cause of cancer death worldwide. In 2002, f1.4 million people worldwide developed gastroesophageal cancers and 1.1 million died (2). Esophageal cancer incidence has increased in recent decades, coinciding with a shift in histologic type and primary tumor location. Adenocarcinoma of the esophagus is now more prevalent than squamous cell carcinoma in the United States and Western Europe, with most tumors located in the distal esophagus (3).The overall 5-year survival rates for gastroesophageal cancers are 20% to 25% despite the aggressiveness of established standard treatments associated with substantial side effects (4, 5). For pancreatic cancer, there is an even greater medical need. Due to the advanced state of the disease at the time of diagnosis, the prognosis is extremely poor, with median survival times of less than 6 months and a 5-year survival rate of <5.5%. The lack of a major benefit from the various newergeneration combination chemotherapy regimens for these cancers has stimulated research into the use of targeted agents, such as small molecule -based kinase inhibitors and monoclonal antibodies (mAb; ref. 6). However, mAbs, such as bevacizumab, cetuximab, and trastuzumab...

Cutaneous T-cell lymphomas (CTCL) are a group of skin neoplasms that originate from T lymphocytes and are difficult to treat in advanced stages. The present study is aimed at the identification of tumor-specific antigens from a human testis cDNA library using human sera known as the SEREX (serological identification of recombinantly expressed genes) approach. A cDNA library from normal testicle tissue was prepared and approximately 2 million recombinants were screened with sera from Sézary Syndrome and Mycosis fungoides patients. A total of 28 positive clones belonging to 15 different genes/ORFs were identified, including five hitherto unknown sequences. Whereas control sera did not react with most clones, 11–71% sera from CTCL patients were reactive against the identified clones. Expression analysis on 28 normal control and 17 CTCL tissues by reverse transcription–PCR (RT-PCR) and Northern blotting revealed seven ubiquitously distributed antigens, six differentially expressed antigens (several normal tissues were positive), and two tumor-specific antigens that were expressed only in testis and tumor tissues: ( i ) A SCP-1-like sequence, which has already been detected in various tumors, has been found in one CTCL tumor and four sera of CTCL patients reacted with various SCP-1-like clones and ( ii ) a new sequence named cTAGE-1 (CTCL-associated antigen 1) was detected in 35% of CTCL tumor tissues and sera of 6/18 patients reacted with this clone. The present study unravels CTCL-associated antigens independent of the T-cell receptor. The SCP-1-like gene and cTAGE-1 were shown to be immunogenic and immunologically tumor-specific and may therefore be candidates for immunotherapy targeting CTCL.

Tumor-associated antigens (TAA) are increasingly used as specific targets for immune therapy of malignant melanoma. The aim of the present study was to provide a basis for selecting the most suitable TAA by analyzing the mRNA expression of a large panel of TAA by RT-PCR and Northern blotting. We have chosen primers differentiating four groups of TAA (MAGE-A, MAGE-B, and two groups of GAGE-genes) and 13 individual TAA (MAGE-A2 and -A3, RAGE-1, -2, -3, and -4, LAGE-1a and -1b, NY-ESO-1, GAGE-1, SSX-2, SCP-1, and cTAGE-1) based on most recent sequence data. In addition, the RAGE-gene family has been separated into its four members by a novel designed nested PCR, which was confirmed by Northern analysis. Furthermore, the chromosomal organization and relationship between the RAGE-family and MOK was analyzed. RAGE-4 mRNA could be shown for the first time to be present in testis tissue. The most frequently expressed TAA were the MAGE-A and the GAGE-3,-4,-5,-6,-8 group, whereas among individual TAA MAGE-A2, -A3, RAGE-1, -3, and LAGE-1a/b were found within most specimens and are thus promising candidates for immune therapy. In comparison, melanoma metastatic specimens and cell lines show similar profiles of TAA expression, but individual TAA differ notably between both types of samples indicating that results from cell lines are not always applicable to tumor specimen.

We have identified a new gene, gbp-5, with high homology to the guanylate binding proteins (GBP) belonging to the GTPase superfamily including the ras gene. gbp-5 is transcribed at least into three splicing variants (gbp-5a, -5b, and -5ta) leading to two different proteins (GBP-5a/b, GBP-5ta). GBP-5ta is C-terminally truncated by 97aa and has therefore lost its isoprenylation site. Although RT-PCR results indicated expression of GBP-5 members in selected normal tissues, western blotting using two newly generated antibodies revealed that expression of both proteins is restricted to peripheral blood monocytes with GBP-5ta at lower levels. In contrast, cutaneous T-cell lymphoma (CTCL) tumor tissues (seven of seven) were positive solely for GBP-5ta, and four of four CTCL cell lines expressed both proteins. Eight of nine melanoma cell lines expressed GBP-5a/b and four of nine additionally low levels of GBP-5ta. SEREX retesting using CTCL sera indicated a higher immunogenicity for GBP-5ta (nine of 16) than for GBP-5a/b (two of 11). Treatment of CTCL cell lines with interferon-gamma did not alter protein expression of GBP-5ta or GBP-5a/b. The restricted expression pattern of both GBP-5ta and GBP-5a/b and the pivotal role of many known members of the GTP-binding proteins in proliferation and differentiation suggest possible cancer-related functions of gbp-5.

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of extra-nodal non-Hodgkin lymphomas with primary manifestation in the skin with poor treatment options in the advanced stages. As basis for future immune-therapeutic strategies we have investigated the possible expression of tumor-specific targets in CTCL focusing mainly on so-called cancer-germline genes. cDNAs derived from 20 CTCL tissues and 4 CTCL cell lines were tested with 15 gene-specific and 4 gene family-specific primers by RT-PCR and confirmative Northern blotting. The most frequently detected mRNAs were LAGE-1 (55% with only partial coexpression of the splicing variants), cTAGE-1 (35%), MAGE-A9 (27%) and the GAGE-3-7 group (35%). Furthermore, we could detect NY-ESO-1 (21%) and a MAGE-A subgroup (15%), whereas subspecification of the latter proved absence of MAGE-A1, -A2, -A3, -A6 and -A12. SCP-1 was found in only one specimen and a several antigens could not been detected in any tumor tissue or cell line (MAGE-B, GAGE-1,2,8 and all 4 RAGE genes). 90% of all CTCL samples were positive for at least 1 of the frequent mRNAs in RT-PCR (LAGE-1, NY-ESO-1, cTAGE-1, MAGE-A9, or GAGE-3to7). Using a secondary SEREX approach we could detect sero-reactivity in sera of CTCL patients against recombinant cTAGE-1 (10/29), GAGE (3/19), MAGE-A1 (1/18), -A3 (1/18), -A6 (2/18) and -A9 (4/18) protein, but not against LAGE-1a, MAGE-A4b or MAGE-A12 protein (n ‫؍‬ 19). We conclude, that certain cancer-germline genes can be detected frequently in CTCL and are able to elicit a systemic immune response. These candidate genes might therefore be promising targets for immunotherapeutic interventions in CTCL.

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cTAGE-1 is a cutaneous-T-cell-lymphoma-specific tumor antigen recently identified by serologic identification of antigens by recombinant expression cloning. This study was aimed at identifying and characterizing related genes. Rapid amplification of cDNA ends and DNA screening led to five new members of the cTAGE gene family belonging to four different genes, two of which were differentially spliced (cTAGE-1/2 and cTAGE-5). Expression analysis using reverse transcription polymerase chain reaction revealed that cTAGE-1, cTAGE-1B, and cTAGE-5A expression was restricted to testis and tumor tissues, whereas the other cTAGE members were found in two to eight other normal tissues (of 27 tissues tested). Tumor-specific protein expression of cTAGE-5 was confirmed by Western blotting. Sero-reactivity against cTAGE-1, cTAGE-4, cTAGE-5A, and cTAGE-5B was found only in tumor patients (cutaneous T cell lymphoma and melanoma). The immunogenic epitope of cTAGE-1 was determined by using epitope mapping and sera of two cutaneous T cell lymphoma patients. Moreover, cTAGE-1, cTAGE-4, cTAGE-5A, and cTAGE-5B could be detected in most types of tumor tissues and cell lines at variable frequencies, including those of cutaneous T cell lymphoma, melanoma, head and neck squamous cell carcinoma, breast carcinoma, and colon carcinoma. We conclude that cTAGE-1 and cTAGE-5 are new cancer germline antigens and that tumor-specific splicing of cTAGE genes may lead to further candidate proteins for specific immunotherapy of cutaneous T cell lymphoma and other malignancies.

Antibody recognition of specific tumour antigens by patients' sera may be used for evaluating the possible immunogenicity of new antigens; serological tests could be used for tumour monitoring purposes.