Serological identification of breast cancer-related antigens from aSaccharomyces cerevisiae surface display library

Abstract

A yeast cell surface display technology was used for the isolation and characterization of tumor antigens recognised by autologous or allogeneic breast cancer serum. More than 100 clones recognized by patient serum were isolated using high-through-put fluorescence activated cell sorting. Combined serological and sequence analysis confirmed that a number of proteins known to be overexpressed in breast cancer tissue could be detected. A recently identified small breast epithelial mucin almost exclusively expressed in mammary gland tissue was isolated as a mutated protein variant. Subsequent serological analysis using the yeast expression system for the wild-type and mutant form showed a strong recognition by patient sera, whereas no significant recognition was observed for the respective prokaryotically expressed proteins. The small breast epithelial mucin is present to a large extent in a membrane bound format and might be used for tumor targeting strategies. ' 2005 Wiley-Liss, Inc.Key words: tumor antigens; serological screening; yeast surface display; cDNA expression library One of the major goals in the field of cancer immunotherapy is the identification of appropriate target antigens for either vaccine or antibody based approaches. The systematic search for tumorspecific antigens and their derived peptides started with the work of Boon et al.1 using T cell epitope cloning and Rammensee et al.2 using peptide elution. The value of both methods has been demonstrated in many areas including cancer. Both methods do have their intrinsic limitations, however, by relying either on the existence of a T-cell line or clone for the epitope cloning approach or on a sufficient amount of tumor tissue to allow for the successful identification of immunogenic peptides. The advent of a serological approach such as the serological identification of antigens by recombinant expression cloning (SEREX) with high-titered immunoglobulin G antibodies obtained from the sera of cancer patients represented a hallmark in modern tumor serology as it allowed for the immediate identification of tumor related antigens.3 SEREX has led to the identification of a multitude of new cancer antigens in different tumor entities and the international SEREX database (www2.licr.org/CancerImmunomeDB/) holds more than 2,000 different gene entries of potentially cancer related antigens. The importance of this technology goes beyond the serological identification of candidate tumor antigens by creating a link between T and B cell immunology as strong antibody responses are considered to depend on the presence of T cell help.4,5 The analysis of the B cell repertoire helps to identify antigens that might become valid targets for immunotherapy with cancer vaccines aiming to induce cytotoxic T cell responses.6,7 Evidence has accumulated over the recent years, however, that the antigen repertoire detectable by the conventional SEREX approach is limited. 8 This might be attributed in part to the fact that potential antigens that are subject to posttranslational ...

Recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant induces broad antibody responses in humans, a RAYS-based analysis

“…When the ratio of the mean fluorescence intensity (g-mean) of CT-antigen expressing yeast versus empty vector expressing yeast was >2, a serum was considered positive. This cut-off point was successfully established in our previous studies (6,9,10) as well as by other groups (12). A positive serum reactivity is shown in Fig.…”

“…As a consequence, we established a eukaryotic cDNA expression system in yeast (RAYS) (6) with the advantage that recombinant proteins can be expressed on the yeast surface as part of the cell wall in a more naturally folded and partially glycosylated manner (7,8). We and others successfully used this system for serological tumor antigen characterization (6,(9)(10)(11)(12).…”

Serological immune response to cancer testis antigens in patients with pancreatic cancer

“…As a consequence, we recently established a eukaryotic cDNA expression system in yeast (RAYS) to overcome this SEREX-inherent problem. 15,16 Yeast has the advantage that recombinant proteins can be expressed on the cell surface as part of the cell wall in a more naturally folded and partially glycosylated manner. [16][17][18] The ability of the yeast display system to detect potentially relevant tumor antigens not recognized by conventional SEREX demonstrates the power of this new tool.…”

“…[16][17][18] The ability of the yeast display system to detect potentially relevant tumor antigens not recognized by conventional SEREX demonstrates the power of this new tool. 15 Considering these facts, it seems to be promising to use SEREX and/or RAYS in combination with a cDNA expression library of pancreatic adenocarcinoma tissue to identify novel pancreas carcinoma-associated antigens. The prerequisite for such an approach would be a sufficiently strong humoral immune response against pancreatic carcinoma antigens.…”

Cryptic Epitopes Induce High-Titer Humoral Immune Response in Patients with Cancer

“…We previously reported the use of rAg expressed on yeast surface technology for the discovery of novel Ags associated with breast cancer (16) and for the detection of autoantibodies to known cancer-testis Ags in patients with pancreatic cancer (17).…”