GD2-MEDIATED MELANOMA CELL TARGETING AND CYTOTOXICITY OF LIPOSOME-ENTRAPPED FENRETINIDE Gabriella PAGNAN 1 , Paolo G. MONTALDO 1 , Fabio PASTORINO 1 , Lizzia RAFFAGHELLO 1 , Marc KIRCHMEIER 2 , Theresa M. ALLEN 2 and Mirco PONZONI 1 * 1 Laboratory of Oncology, G. Gaslini Children’s Hospital, Genoa, Italy 2 Department of Pharmacology, University of Alberta, Edmonton, Canada Melanoma is a highly malignant and increasingly common neoplasm. Because metastatic melanoma remains incurable, new treatment approaches are needed. Immunoliposomes have been previously shown to enhance the selective localiza- tion of immunoliposome-entrapped drugs to solid tumors with improvements in the therapeutic index of the drugs. Previously, we reported that the synthetic retinoid fenret- inide (H PR) is an inducer of apoptosis in neuroblastoma (N B) cells, sharing the neuroectodermal origin with melanoma cells. HPR is a strong inducer of apoptosis also in melanoma cells, although at doses 10-fold higher than those achievable clinically. Thus, our purpose was to investigate the in vitro potentiation of its cytotoxic effect on melanoma cells in combination with long-circulating GD2-targeted immunolipo- somes. GD2 is a disialoganglioside extensively expressed on tumors of neuroectodermal origin, including melanoma. Mu- rine anti-GD2 antibody (Ab) 14.G2a and its human/mouse chimeric variant ch14.18 have been ligated to sterically stabilized liposomesby covalent coupling of Ab to the polyeth- ylene glycol (PEG) terminus. Ab-bearing liposomes showed specific, competitive binding to and uptake by various mela- noma cell lines compared with liposomes bearing non- specific isotype-matched Abs or Ab-free liposomes. Cytotox- icity wasevaluated after 2 hr treatment, followed by extensive washing and 72 hr incubation. This treatment protocol was designed to minimize non-specific adsorption of liposomes to the cells, while allowing for maximum Ab-mediated binding. W hen melanoma cells were incubated with 30 M HPR entrapped in anti-GD2 liposomes, a significant reduction in cellular growth was observed compared to free HPR, en- trapped HPR in Ab-free liposomes or empty liposomes. Cytotoxicity was not evident in tumor cell lines of other origins that did not express GD2. Growth of N B cells was also inhibited by immunoliposomes with entrapped H PR. Int. J. Cancer 81:268–274, 1999.  1999 Wiley-Liss, Inc. Melanoma is becoming a rather common disease. In the past decade, the incidence of melanoma increased faster than that of any other solid malignancy (Balch et al., 1989; Marshall, 1998). Since metastatic or advanced-stage melanoma (Ketcham and Balch, 1985) is refractory to conventional treatment such as chemotherapy and radiation therapy, novel therapeutic approaches are required. Retinoids are a class of natural or synthetic compounds, structurally related to vitamin A, which participate in the control of cell proliferation, differentiation and fetal development. The syn- thetic retinoid N-(4-hydroxyphenyl)retinamide (HPR; fenretinide) inhibits carcinogenesis in animal models for cancers of the breast, bladder, lung, ovary and prostate (Pollard et al., 1991; Formelli et al., 1996). Various clinical chemoprevention trials focused on cancers of the breast, prostate, cervix, skin and lung are ongoing (Formelli et al., 1996; Kelloff, 1994). In addition, HPR has exhibited therapeutic effects by causing tumor regression in animals bearing carcinogen-induced or xenotransplanted human tumors (reviewed by Kelloff, 1994). Reports that HPR can induce apoptosis in a variety of tumor cell types (reviewed by Lotan, 1995) suggest that this activity may be important for its chemopreventive and therapeutic effects. Previously, we reported (Ponzoni et al., 1995) that the induction of programmed cell death by HPR also inhibits growth of human neuroblastoma (NB) cells that are of neuroectodermal origin like melanoma cells. On the basis of these findings, we evaluated its in vitro effects on human melanoma cell lines. We found that HPR is a potent inducer of apoptosis in melanoma cells (Montaldo et al., 1999), albeit at concentrations 2- to 10-fold higher than those clinically achievable (Formelli et al., 1993, 1998). We therefore studied a system of sterically stabilized immunoliposomes in an effort to significantly increase the selective localization of HPR to tumor cells and to increase the half-life and stability of this promising anticancer agent. Such antibody (Ab)-directed liposomes have been recog- nized as an efficient tool for the site-specific delivery of drugs and diagnostic agents. However, the in vivo use of classical immunoli- posomes is hampered by their very rapid clearance from the circulation by the reticuloendothelial system (RES) (Allen, 1994; Lasic, 1996). Incorporation of polyethylene glycol (PEG)- derivatized phospholipids into liposomes results in carriers that can enhance the therapeutic efficacy of encapsulated drugs by impart- ing the ability to escape the RES and remain in the circulation for prolonged periods of time (Allen, 1994). Ab-based targeting is a promising approach in the development of cancer therapies. Among various antigens found on malignant cells, disialoganglioside GD2 is an attractive target for therapy (Frost et al., 1997; Uttenreuther-Fischer et al., 1995), since it is extensively expressed on tumors of neuroectodermal origin, such as NB, melanoma and to a lesser extent on small-cell lung carcinoma, glioma and some sarcomas (Mujoo et al., 1986; Schulz et al., 1984). The relatively tumor-specific expression of GD2 and its restricted presence in normal tissues such as the peripheral and central nervous system, the latter protected from liposomes and immunoglobulins by the blood-brain barrier, make GD2 a suitable target for Ab-mediated, liposome-based therapy of neuroectoderma- derived tumors. In this report, we show that HPR delivered by stabilized GD2-mediated immunoliposomes is very effective in blocking melanoma cell proliferation in vitro. MATERIAL AND METHODS Chemicals HPR and N-(4-methoxyphenyl)retinamide (MPR) (kindly sup- plied by Johnson Pharmaceutical Research Institute, Spring House, PA) were dissolved in absolute ethanol at 10 mmol/l and stored in aliquots at -20°C (up to 1 week). Hydrogenated soy phosphatidyl- choline (HSPC), cholesterol (CH), 1,2 distearoylglycero-3- phosphatidylethanolamine-N-polyethylene glycol-2000 (DSPE- PEG) and rhodamine phosphatidylethanolamine (rhoda-PE) were from Avanti Polar Lipids (Alabaster, AL). The PEG derivative of PE modified with a maleimide group at the distal terminus of the Grant sponsors: Associazione Italiana Ricerca sul Cancro (A.I.R.C.), Associazione Italiana Neuroblastoma and Ricerca Corrente Ministeriale Gaslini. *Correspondence to: Laboratory of Oncology, G. Gaslini Children’s Hospital, Largo G. Gaslini 5, I-16148, Genoa, Italy. Fax: (39)010-3779820. E-mail: molbiol@tin.it. Received 31 July 1998; Revised 11 November 1998 Int. J. Cancer: 81, 268–274 (1999)  1999 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer