Development of Fab′ fragments of anti-GD2 immunoliposomes entrapping doxorubicin for experimental therapy of human neuroblastoma

doi:10.1016/S0304-3835(03)00099-5

Cancer Letters

Volume 197, Issues 1–2, 18 July 2003, Pages 199-204

https://doi.org/10.1016/S0304-3835(03)00099-5 Get rights and content

Abstract

Neuroblastoma (NB) is the most common extra-cranial solid tumor in children. Since intensive therapeutic intervention does not prolong the overall disease-free survival rate for this tumor, novel therapeutic strategies are required. NB tumor, but not normal tissues, over-express the disialoganglioside (GD₂) at the cell surface. In this study we developed a novel immunoliposomal formulation by covalently coupled Fab′ fragments of the monoclonal antibody anti-GD₂ to Stealth liposomes (Fab′-SIL). In vitro experiments showed specific, competitive binding to, and uptake by various NB cell lines. Moreover, doxorubicin-loaded immunoliposomes (Fab′-SIL[DXR]) presented increased selectivity and efficacy in inhibiting NB cell proliferation compared to free drug and non-targeted liposomes (SL[DXR]). The in vivo cytotoxic effectiveness of different liposomal formulations encapsulating DXR was tested against an experimental metastatic model of human NB in nude mice. Long term survivors were obtained in mice treated with Fab′-SIL[DXR], but not in untreated animals or those treated with free anti-GD₂ Fab′ fragments, Fab′-SIL (no drug), free-DXR or SL[DXR] (P<0.0001). Fab′-SIL[DXR] prevented the establishment and the metastatic growth of the tumor cells in all organs examined. In conclusion, Fab′-SIL[DXR] formulations should receive clinical evaluation as adjuvant therapy of neuroblastoma.

Introduction

The effective treatment of neuroblastoma (NB) remains one of the major challenges in pediatric oncology. The use of intensive therapeutic interventions have only marginally prolonged the overall long-term disease-free survival rates, mainly due to the dose-limiting toxicity associated with systemic delivery of cytotoxic drugs in vivo [1]. Innovative experimental therapies are thus required to eradicate residual disease after chemotherapy and surgery.

NB is a chemosensitive tumor and cytotoxic agents such as doxorubicin (DXR) are considered to be important and effective treatment modalities. However, the therapeutic efficacy of this anticancer drug is restricted by dose-limiting toxicity to bone marrow and heart tissue [2]. The selective toxicity of DXR would be greatly improved if the concentration of drug in tumors could be increased relative to that in sensitive normal tissues.

In the last few years long circulating (sterically stabilized or Stealth) liposomal formulation of doxorubicin (Doxil/Caelyx) has been approved for use in the treatment of both Kaposi's sarcoma and ovarian cancer [3], [4]. In patients receiving large cumulative doses of doxorubicin, clinical cardiotoxicity was reduced [5] and this liposomal formulation of doxorubicin is now under advanced clinical trial evaluation for several human solid cancers.

Several studies have shown that the use of immunoliposomes (SIL) significantly increased target cell binding in vitro [6], [7], reduced toxicity and improved therapeutic efficacy in vivo [8]. Moreover, the coupling of antibody Fab′ fragments instead of whole immunoglobulin molecules to Stealth liposomes have increased their circulation times [9] and improved their in vivo efficacy [10]. Since NB tumors expresses abundant amounts of the disialoganglioside GD₂ at the cell surface, and since this internalizing epitope is only minimally expressed by normal tissues [11], the use of Fab′ fragments of anti-GD₂, as a ligand for targeting liposomal doxorubicin to NB, merits further investigation.

In this study we examined the specific binding of anti-GD₂-targeted liposomal formulations against GD₂-positive NB cell lines in vitro; selective cytotoxicity of these DXR-loaded immunoliposomes and survival times were evaluated in nude mice xenografts of the human NB cell line, HTLA-230. The HTLA-230 cells, when administered intravenously in nude mice, provides an in vivo experimental model for the metastatic spread of tumor in human disease [12]. Our results clearly show that liposomal DXR targeted via Fab′ fragments of anti-GD₂ were effective in suppressing the growth of metastases in mice implanted with a human neuroblastoma tumor.

Section snippets

Characterization of Fab′ fragments of anti-GD₂ immunoliposomes

Liposomes were prepared by repeated extrusion through polycarbonate filter membranes with a pore size of 100 nm and DXR was entrapped into liposomes via an ammonium sulfate gradient as previously reported [13]. The loading efficiency of DXR was greater than 95% and liposomes routinely contained DXR at a concentration of 150–180 μg DXR/μmol PL.

Previous preclinical studies showed that lysis of NB cells by mouse and chimeric anti-GD₂ resulted from antibody-dependent cellular cytotoxicity and

Discussion

A number of studies conducted on large cohorts of patients have shown that the presence of circulating neuroblastoma cells in the blood and micrometastases in the bone marrow at the time of primary surgery are a strong predictors of relapse [16]. Since bone marrow micrometastases are a direct measurement of the ability of tumor cells to spread systemically, the establishment of a model that closely mimics the clinical situation allows a more realistic evaluation of antitumor therapies. Thus,

Acknowledgements

We thank Dr R.A. Reisfeld for providing us with the hybridoma 14G.2a secreting a murine mAb of immunoglobulin (Ig) G2a isotype subclass, specific for the GD₂ antigen and Puja Sapra for expert technical assistance. F. P. is a recipient of a Fondazione Italiana per la lotta al Neuroblastoma fellowship. Work supported by Fondazione Italiana per la lotta al Neuroblastoma, Costa Crociere, and Associazione Italiana Ricerca Cancro (AIRC).

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Mini-reviewDevelopment of Fab′ fragments of anti-GD2 immunoliposomes entrapping doxorubicin for experimental therapy of human neuroblastoma

Abstract

Introduction

Section snippets

Characterization of Fab′ fragments of anti-GD2 immunoliposomes

Discussion

Acknowledgements

J. Controlled Release

FEBS Lett.

Multivariate analysis of risk factors in stage 4 neuroblastoma patients over the age of 1 year treated with megatherapy and stem-cell transplantation: a report from the European Bone Marrow Transplantation Solid Tumor Registry

J. Clin. Oncol.

Anthracyclines in hematology: preclinical studies, toxicity and delivery systems

Blood Rev.

Doxorubicin encapsulated in liposomes containing surface-bound polyethylene glycol: pharmacokinetics, tumor localization, and safety in patients with AIDS-related Kaposi's sarcoma

J. Clin. Pharmacol.

Phase II study of liposomal doxorubicin in refractory ovarian cancer: antitumor activity and toxicity modification by liposomal encapsulation

J. Clin. Oncol.

Pegylated liposomal formulation of doxorubicin (doxil): reduced clinical cardiotoxicity in patients reaching or exceeding cumulative doses of 500 mg/m2

Ann. Oncol.

Antibody-mediated specific binding and cytotoxicity of liposome-entrapped doxorubicin to lung cancer cells in vitro

Cancer Res.

Development of anti-p185HER2 immunoliposomes for cancer therapy

Proc. Natl. Acad. Sci. USA

Mini-review
Development of Fab′ fragments of anti-GD₂ immunoliposomes entrapping doxorubicin for experimental therapy of human neuroblastoma

Characterization of Fab′ fragments of anti-GD₂ immunoliposomes

Pegylated liposomal formulation of doxorubicin (doxil): reduced clinical cardiotoxicity in patients reaching or exceeding cumulative doses of 500 mg/m²

Development of anti-p185^HER2 immunoliposomes for cancer therapy