Stable GM 3 Lactone Mimetic Raises Antibodies Specific for the Antigens Expressed on Melanoma Cells Annarosa Arcangeli,* ,† Lucio Toma,* ,‡ Luca Contiero, § Olivia Crociani, † Laura Legnani, ‡ Carlotta Lunghi, § Elisa Nesti, † Gloriano Moneti, | Barbara Richichi, § and Cristina Nativi* ,§,⊥ Dipartimento di Patologia e Oncologia Sperimentali, Universita’ di Firenze, Firenze, Italy, Dipartimento di Chimica Organica, Universita’ di Pavia, Pavia, Italy, Dipartimento di Chimica, Universita’ di Firenze, Firenze, Italy, Dipartimento di Farmacologia, Universita’ di Firenze, Firenze, Italy, and FiorGen, Universita’ di Firenze, Firenze, Italy. Received December 17, 2009; Revised Manuscript Received June 23, 2010 Immunotherapy of tumors and of melanoma in particular has a long history, and recently this therapeutic approach found a reliable scientific rationale. This biological therapy aims to teach the patient’s immune system to recognize the antigens expressed on tumor cells and destroy them, leaving normal cells intact. The success of this therapy highly depends on the selection of target antigens that are essential for tumors growth and progression. The overexpression of GM 3 ganglioside 1 and especially the expression of its metabolite GM 3 lactone 2 characterize murine and human melanomas, playing an important role in tumor progression and making such self-antigens potential targets for the immunotherapy of these neoplasms. Although more immunogenic than its precursor, GM 3 lactone 2 is unsuitable to be used in immunotherapy as a melanoma-associated antigen (MAA) because it is unstable under physiological conditions. We designed and synthesized the hydrolytically stable mimetic 3, which is remarkably simpler than the native lactone 2; after conjugation of 3 to the protein carrier keyhole-limpet hemocyanin (KLH), the obtained glycoprotein 5 was used as the immunogen in ViVo to successfully elicit specific antimelanoma antibodies. In fact, no appreciable binding to GM 1 was observed. Capitalizing on the stability and on the reduced structural complexity of mimetic 3, the immunostimulant 5 we report represents a new promising synthetic glycoconjugate for the immunotherapy of melanoma. INTRODUCTION Oncogenic transformations are accompanied by biochemical and functional changes. In particular, qualitative and quantitative changes in carbohydrate composition of the glycocalix during cell differentiation and proliferation have been observed and seem to reflect the state of malignant transformation of a variety of human tumors (1-4). Although the biological significance of such changes has not been elucidated yet, it is widely accepted that they contribute to or trigger an uncontrolled cellular adhesion, making the cell potentially metastatic. In recent years, a variety of monoclonal antibodies have been developed to specifically recognize carbohydrate epitopes that result from these faulty glycosylation processes. These epitopes (5-8), known as tumor associated antigens (TAAs), have been suc- cessfully used as markers of tumor progression and include carbohydrates expressed on normal tissues but which are accumulated in high density on the surface of tumor cells (5). Gangliosides are sialic acid containing glycosphingolipids composed of a complex carbohydrate moiety linked to a hydrophobic ceramide portion. Embedded within the outer leaflet of the cell membrane, the carbohydrate chain is exposed to the extracellular matrix. GM 3 ganglioside 1 (Figure 1), a glycosph- ingolipid found in essentially all types of cells and tissues, is the major ganglioside in normal melanocytes and is overex- pressed in melanoma cells with metastatic potential (9, 10). The corresponding GM 3 lactone 2 (Figure 1) has also been found in melanoma as a minor component (11, 12); its formation is likely promoted by the lower pH environment of tumor cells (13) and, possibly, by a different conformation of GM 3 ganglioside induced on the tumor cell surface as a result of its local high density (12). GM 3 ganglioside has been widely investigated as a potential vaccine against cancer, although with scarce success since the majority of melanoma associated antigens (MAAs) are weakly immunogenic, owing to their nature and tissue distribution (14). Nevertheless, as metastatic melanoma appears to be resistant even to the most recent molecularly targeted agents, immunotherapeutic strategies continue to be used in an attempt to improve the results of the last 10 years of clinical investigations both in active and adoptive immune therapy. Indeed, vaccines prepared by using specific tumor antigens have recently lead to the commercial availability of the first tumor immunotherapeutic agents, which are usually employed in the postsurgery treatment of removed tumors, to stimulate the patient to develop the appropriate immune defenses, in order to inhibit the recruitment of the same tumor and avoid far more invasive chemo- or radiotherapeutic agents (12, 15). To date, a large repertoire of MAAs are available, most of which are extensively studied for their possible use in immunotherapy (16). Recent findings showing relevant clinical responses in metastatic melanoma patients proved the principle that, under appropriate conditions, even large tumor masses can be made to regress by immune interventions (17). Although more immunogenic than GM 3 ganglioside 1, GM 3 lactone 2 (Figure 1) also failed as an immunostimulant because, under physiological conditions, the amount of lactone is below * To whom correspondence should be addressed. Prof. A. Arcangeli: Tel, +39 0554598206; Fax, +39 0554598900; E-mail, annarosa.arcangeli@ unifi.it. Prof. L. Toma: Tel, +39 0382987843; Fax, +39 0382987323; E-mail, lucio.toma@unipv.it. Prof. C. Nativi: Tel, +39 0554573540, Fax, +39 0554573570; E-mail, cristina.nativi@unifi.it. † Dipartimento di Patologia e Oncologia Sperimentali, Universita’ di Firenze. ‡ Universita’ di Pavia. § Dipartimento di Chimica, Universita’ di Firenze. | Dipartimento di Farmacologia, Universita’ di Firenze. ⊥ FiorGen, Universita’ di Firenze. Bioconjugate Chem. 2010, 21, 1432–1438 1432 10.1021/bc900557v  2010 American Chemical Society Published on Web 07/29/2010