Cancer Letters 197 (2003) 151–155 www.elsevier.com/locate/canlet
Immunoliposomal fenretinide: a novel antitumoral drug for human neuroblastoma L. Raffaghelloa,*, G. Pagnana, F. Pastorinoa, E. Cosimoa, C. Brignolea, D. Marimpietria, E. Bogenmannb, M. Ponzonia,c, P.G. Montaldoa a
Laboratory of Oncology, G. Gaslini Children’s Hospital, Largo G. Gaslini 5, 16148 Genoa, Italy Laboratory of Hematology/Oncology, Children’s Hospital, University of South California, Los Angeles, CA, USA c Differentiation Therapy Unit, Department of Hematology/Oncology, G. Gaslini Children’s Hospital, Genoa, Italy
b
Received 25 November 2002; accepted 29 November 2002
Abstract Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In advanced disease stages, prognosis is poor and treatments have limited efficacy, thus novel strategies are warranted. The synthetic retinoid fenretinide (HPR) induces apoptosis in NB and melanoma cell lines. We reported an in vitro potentiation of HPR effects on melanoma cells when the drug is incorporated into GD2-targeted immunoliposomes (anti-GD2-SIL-HPR). Here, we investigated the antitumor activity of antiGD2-SIL-HPR against NB cells, both in vitro and in vivo. Anti-GD2-immunoliposomes (anti-GD2-SIL) showed specific, competitive binding to, and uptake by, various NB cell lines. Moreover, anti-GD2-SIL-HPR presented increased selectivity and efficacy in inhibiting NB cell proliferation through the induction of apoptosis, compared to free drug and SL-HPR. In an in vivo NB metastatic model, we demonstrated that anti-GD2-SIL-HPR completely inhibited the development of macroscopic and microscopic metastases in comparison to controls. However, similar, but significantly less potent antitumor effect was observed also in mice treated with anti-GD2 immunoliposomes without HPR (anti-GD2-SIL-blank) or anti-GD2 mAb alone (P ¼ 0:0297 and P ¼ 0:0294, respectively, vs. anti-GD2-SIL-HPR). Moreover, our results clearly demonstrated that, although anti-GD2 mAb had a strong antitumor effect in this in vivo NB model, 100% curability was obtained only following treatment with antiGD2-SIL-HPR (P , 0:0001). Anti-GD2 liposomal HPR should receive clinical evaluation as adjuvant therapy of neuroblastoma. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Liposomes; Neuroblastoma; Fenretinide; Apoptosis; Anti-GD2; Metastasis; Immunolipsomes; Adjuvant therapy; Retinoid
1. Introduction Neuroblastoma (NB) is the most common solid * Corresponding author. Tel.: þ39-010-5636342; fax: þ 39-0103779820. E-mail address:
[email protected] (L. Raffaghello).
tumor of childhood, derived from the sympathetic nervous system [1]. Despite the favorable prognosis in children with localized NB, the poor clinical outcome and low response to conventional therapy in patients with advanced stage disease require novel therapeutic approaches. Recently, several new strategies have emerged to circumvent such treatment failures. Much
0304-3835/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3835(03)00097-1
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work has recently focused on the possibility of more selectively triggering the pathways of programmed cell death [2]. A promising discovery in this field is fenretinide (HPR), a synthetic retinoid related to vitamin A, which has been shown to inhibit the growth of different tumor cell lines, including NB [3]. HPR is currently in Phase I and II clinical trials in NB patients. So far, maximum peak plasma concentrations of HPR of 1 – 3 mM have been achieved, and at this drug concentration several NB cell lines were recently shown to be partially resistant in vitro [4,5]. From the above premises, it appears clear that an improvement in the effectiveness of HPR treatment in vivo would strongly benefit from a specific and selective targeting of the apoptotic agent to tumor cells. To enhance the stability and the specific tumor localization of HPR, we explored a system of sterically stabilized immunoliposomes (SIL). The advantage of encapsulating cytotoxic drugs in SIL over free drug administration has been demonstrated in a number of early (micrometastatic) solid tumor models [6]. The specificity of stabilized liposomes (SL) is supported by coupling specific ligands, e.g. a specific tumor antibody, to the outer surface of the lipid bilayer. Disialoganglioside GD2 is abundantly expressed in neuroectoderma-derived tumors and minimally in normal tissues such as peripheral nerves and cerebellum [7]. Hence GD2 is an ideal targeting ligand for NB cells. Here, we report that anti-GD2-SIL-HPR show increased selectivity and efficacy in inducing growth arrest of NB cells in vitro through the induction of apoptosis, compared to free drug and SL-HPR. Moreover, we show that anti-GD2-SIL-HPR completely inhibited the development of macroscopic and microscopic metastases, resulting in a significant increase of long term survival in an in vivo human NB metastatic model.
2. Characterization of HPR entrapped in antiGD2-immunoliposomes Liposomes were prepared by repeated extrusion through polycarbonate filter membranes with a pore size of 100 nm, as previously described [4,8]. HPR was entrapped in liposomes with greater than 75% trapping efficiency [4,8].
2.1. Binding of anti-GD2-immunoliposomes Binding and uptake of anti-GD2-SIL at 100 nmol or 400 nmol PL/ml was < 10 or 20-fold higher, respectively, than that obtained with non targeted liposomes. In competition experiments, we observed that the binding of anti-GD2-SIL to GD2-expressing NB cells was completely abolished by the presence of 20-fold excess of free anti-GD2 mAb: conversely, an excess of free non specific Ab did not reduce the binding rate of anti-GD2-SIL These results indicate that the enhanced association of GD2-targeted liposomes to NB cells is due to the antibody-mediated specific recognition of GD2 molecules. 2.2. Uptake of HPR entrapped in anti-GD2immunoliposomes Uptake of GD2-targeted immunoliposomes (antiGD2-SIL-HPR) appeared to saturate within the 2 h incubation period with intracellular concentrations of HPR delivered to NB cells by anti-GD2-SIL reaching about 200 ng/mg of cell protein vs. 110 ng/mg of those obtained by free HPR (Fig. 1A). We also evaluated the rate of decrease of HPR levels from NB cells: although the cellular levels decreased at the same rate for both preparations, the concentration of intracellular HPR was higher after treatment with anti-GD2-SIL-HPR compared to free HPR at any time-point examined (Fig. 1B). Thus, treatment with HPR entrapped in anti-GD2-SIL leads to a considerable increase in the dose per time exposure of cells to HPR compared to free drug.
3. HPR entrapped in anti-GD2-immunoliposomes inhibits NB cell proliferation through the induction of programmed cell death We compared the effects of different HPR formulations on cell proliferation in a panel of NB cell lines expressing different amounts of GD2 and other non-neuroectoderma-derived GD2-negative cells, A431 and HL-60. The cells were treated with different HPR-formulations for 2 h followed by washing and 72 h incubation. Table 1 clearly shows a direct correlation between the extent of GD2 expression and the sensitivity to anti-GD2-SIL-
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Table 1 Sensitivity to anti-GD2-SIL-HPR induced growth arrest and apoptosis of NB cells as function of GD2 expression Cell lines
GD2 expressiona (%)
IC50b (mM)
Apoptotic cellsc (%)
HTLA-230 GI-LI-N ACN IMR-32 A-431 HL-60
97 ^ 3 91 ^ 7 70 ^ 7 57 ^ 6 ,5 ,5
2.5 ^ 0.5 3^1 7.5 ^ 1.5 10.5 ^ 2.5 .50 .50
95 ^ 5 90 ^ 9 75 ^ 7 51 ^ 6 ,1 ,1
a
GD2 expression was evaluated by flow cytometry. IC50 was evaluated as dose-response inhibition of cell count after 2 h of treatment with anti-GD2-SIL-HPR, washing and 3 days of culture. c Cell death was evaluated by [3H]-thymidine labeling of fragmented DNA after 2 h of treatment with 10 mM anii-GD2SIL-HPR, washing and 3 days of culture. The data are means ^ SD of three different experiments. b
Fig. 1. Time course of free HPR or anti-GD2-SIL-HPR uptake (A) and release (B) by neuroblastoma cells in culture. The data presented have been obtained with HTLA-230 cells and are representative of those obtained with other GD2-positive neuroblastoma cell lines, with only negligible differences. (Panel A) Cells were incubated for the indicated times with 5 mM free HPR (K) or anti-GD2-SIL-HPR (W). Then, the cells were washed three times in complete medium, treated with NaCl/0.2 M acetic acid (pH 2.5) and kept at 4 8C for 2 min to eluate surface-bound liposomes. HPR was monitored by HPLC after cell lysis and extraction. (Panel B) cells were incubated for 2 h with the above concentration of free HPR (K) or anti-GD2-SIL-HPR (W), extensively washed, treated with NaCl/0.2 M acetic acid (pH 2.5) and reincubated in drug-free complete medium. At the indicated times, intracellular HPR was monitored by HPLC after cell lysis and extraction. The data presented are means ^ SD of three individual experiments.
HPR-induced growth arrest. Indeed, treatment with anti-GD2-SIL-HPR yielded marked toxicity in GD2expressing cells only, while GD2-negative cell lines were virtually resistant. Noteworthy, NB cells GI-LIN and HTLA-230, roughly 100% GD2-positive, were the most sensitive to the anti-GD2-SIL-HPR treatment, among the cell lines tested. In order to ascertain whether the enhanced cytotoxicity of anti-GD2targeted immunoliposomes depended on specific Ab-mediated recognition, we performed competition
studies. Pretreatment of NB cell lines with 20-fold excess of free anti-GD2 mAb abrogated the antiproliferative effect of anti-GD2-SIL-HPR (data not shown). In order to evaluate if the antiproliferative effect induced by anti-GD2-SIL-HPR was due to the induction of programmed cell death, we used an assay that quantitatively measures the ratio between labeled DNA released into cytosol and culture medium after inter-nucleosomal fragmentation and total DNA labeling [3,9]. Our results clearly show a pronounced induction of apoptosis in NB cell lines following treatment with anti-GD2-SIL-HPR. Such effect was directly correlated to the expression of the GD2 antigen on the cell surface (Table 1). These results have been confirmed by TUNEL assay, flowcytometric evaluation of PI-stained cells and by ultrastructural analysis using electron microscopy. We conclude that anti-GD2-SIL-HPR specifically inhibit cell proliferation of GD2-expressing NB cells through the induction of apoptosis.
4. Anti-tumor activity of anti-GD2-SIL-HPR in an animal model of human experimental metastatic NB These promising in vitro results led us to investigate the antitumor activity of targeted liposo-
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mal formulations of HPR in a human experimental metastatic model of NB, that mimics the in vivo metastatic spread of disease, by intravenous injection of tumor cells [10]. Since bone marrow micrometastases are a direct measurement of the ability of tumor cells to spread systemically, the establishment of a model which closely mimics the clinical situation allows a more realistic evaluation of antitumor therapies. Hence, the metastatic model employed in these studies provides a consistent test for the potential use of targeted liposomal HPR in human metastatic disease. The schedules of treatment were deliberately chosen to allow evaluation of the effects of treatments at different steps of the metastatic cascade, i.e., during the early stages in which tumor cells are in the intravascular circulation and endothelium-attachment take place, and during later stages, when extravasation, stromal invasion and colonization take place as described by others on different models. Our results clearly demonstrate that anti-GD2-SIL-HPR were able to inhibit macro- and micro-metastases at different stages of tumor invasion, leading to significantly prolonged mean survival times; in contrast, SL-HPR and free HPR appeared to be ineffective. Specifically, a long term survival analysis of animals treated with the different regimens has been performed. Mice treated with anti-GD2-SILHPR, anti-GD2-SIL-blank or anti-GD2 mAb alone showed a statistically significant increase life span and mean survival time (Log-Rank test, P , 0:0001). However, anti-GD2-SIL-HPR was significantly more potent in inhibiting metastatic spread of HTLA-230 cells than free anti-GD2 mAb (P ¼ 0:0297) or antiGD2-SIL-blank (P ¼ 0:0294). Specifically, 100% of the mice treated with anti-GD2-SIL-HPR and 80% of the mice treated with anti-GD2-SIL-blank or antiGD2 mAb alone were long-term survivors (Fig. 2). No toxicity at all has been observed in any animal treated with the different HPR formulations. Although the dosage of anti-GD2 mAb used herein is below that so far used in the clinical protocols, our results seem to indicate that anti-GD2 mAb alone is responsible of a great part of the observed antitumor effect; however, it should be pointed out that the 100% of curability has been obtained only with the combination of anti-GD2 immunoliposomes and HPR. Immunotherapy by mAbs can be contributed to by antibody-dependent cell cytotoxicity (ADCC) (including macrophages
Fig. 2. Kaplan–Meier survival curves of CD1 nude/nude mice treated with different liposomal HPR formulations. The animals (n ¼ 10 animals/group) were injected i.v. with 3 £ 106 HTLA230/mouse, and treated after 4 h with different HPR formulations for 5 days. Treatments: control, HEPES Buffer pH 7.4 (A); free HPR, 15 mg/Kg/total dose (W); SL-HPR, 15 mg/Kg/total dose (K);anti-GD2 mAb, 2 mg mAb/Kg/total dose mAb ( 1 ); anti-GD2SIL-blank, 2 mg mAb/Kg/total dose ( £ ); anti-GD2-SIL-HPR, 15 mg HPR/Kg/total dose and 2 mg mAb/Kg/total dose (X). The experiment has been repeated three times with similar results.
and granulocytes), HNK cells activity and complement and the relative contribution of each of these mechanisms is presently under investigation in our lab. Moreover, future in vivo experiments using Fab’ fragments of anti-GD2 coupled to stealth liposomes, no immunogenic toward NB cells, will clarify the therapeutic potential of HPR entrapped in immunoliposomes. The lack of relevant efficacy of immunotherapy with anti-GD2 mAb when used as single agent in NB patients [11], however suggests that, in the clinical setting, immunomediated NB cell killing is relatively inefficient, especially in patients whose ADCC activity is depressed by previous chemotherapy [12]. In this view, our observation of improvement of cell killing, up to a complete cure of animals, when an apoptotic stimulus is added to the immunological challenge, could be of high clinical importance.
5. Conclusions Delivery of anticancer drugs by means of anti-GD2 immunoliposomes seems to be an effective strategy for the treatment of early metastatic disease, when the target cells are resident primarily within the vascu-
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lature. Selective cytotoxicity of anti-GD2-SIL-HPR has been demonstrated in NB cell lines. Moreover, the immunoliposomal HPR formulation, which combines the effect of HPR with that of anti-GD2 mAb, has been proved to be the most efficacious in the treatment of an in vivo experimental metastatic human NB model. Thus, this sinergistic strategy may provide a novel and effective tool for cancer therapy, and may, in particular, be useful for the adjuvant treatment of advanced stage NB.
Acknowledgements We thank Dr. V. Pistoia for helpful discussions and Dr R.A. Reisfeld for kindly providing anti-GD2 14G2a monoclonal antibody. This work has been supported by Italian Association for Cancer Research (A.I.R.C) and Fondazione Italiana per la lotta al Neuroblastoma.
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