Enhanced cytotoxicity of monoclonal anticancer antibody 2C5-modified doxorubicin-loaded PEGylated liposomes against various tumor cell lines

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Abstract

Doxorubicin-loaded long-circulating liposomes (Doxil™, ALZA Corp.) were additionally modified with the nucleosome-specific monoclonal antibody 2C5 (mAb 2C5) recognizing a broad variety of tumor cells via the tumor cell surface-bound nucleosomes. These mAb 2C5-modified PEGylated liposomes demonstrated 3–8-fold increase in the in vitro binding and internalization by multiple cancer cell lines of diverse origins (murine LLC, 4T1, C26 and human BT-20, MCF-7, and PC3), as shown by flow cytometry (FACS) and epi and confocal microscopy. As a result, mAb 2C5-modified Doxil™ demonstrated significantly higher cytotoxicity towards various cancer cells, including those resistant to doxorubicin, than all control preparations. The specific internalization of the mAb 2C5-Doxil™ into cytosol, along with the nuclear localization of their drug load, inside the target cancer cells were mainly responsible the superior anticancer activity. The IC50 values of mAb 2C5-Doxil™ with various murine and human cancer cells were 5–8-fold lower than those of control doxorubicin-loaded liposomes, Doxil™ or Doxil™ modified with a nonspecific IgG.

Introduction

Liposomal formulations of various anticancer drugs are widely used in experimental and clinical oncology with some of them, such as Doxil™, becoming drugs of choice under certain conditions (Alberts et al., 2004, James et al., 1994, Tejada-Berges et al., 2002). The use of the liposomal carrier allows for decreased side effects of cancer chemotherapeutics, such as nonspecific toxicity and for enhanced drug delivery into tumors (Gabizon et al., 2004, Safra et al., 2000). Doxil™ represents doxorubicin incorporated into long-circulating PEGylated liposomes and demonstrates decreased cardiotoxicity and improved tumor accumulation via the enhanced permeability and retention (EPR) mechanism (Gabizon, 1992, Northfelt et al., 1996).

The further development of the concept of long-circulating liposomes involves the attempt to combine the properties of long-circulating liposomes and specifically targeted liposomes in one preparation. The accumulation of the liposomal drugs was shown to be still further improved by their specific targeting to the tumor, that is, by attaching certain tumor-specific molecules to the liposome surface (Sapra et al., 2005, Torchilin, 2005). Specific vector molecules capable of recognizing tumors include antibodies, peptides, folate, transferrin, and some other moieties (Kurohane et al., 2000, Schiffelers et al., 2003, Willis and Forssen, 1998). With this in mind, several attempts have been made to further improve the anticancer efficiency of Doxil™ by actively targeting it to tumors with certain vector molecules specific to the receptors characteristic of cancer cells (Park et al., 2001). On a broader picture, targeted doxorubicin-loaded liposomes were repeatedly shown to demonstrate an increased anticancer activity, as did, for example, folate-targeted liposomes loaded with doxorubicin with target cells in vitro (Lee and Low, 1995). Doxorubicin-loaded liposomes conjugated with folic acid were shown to be internalized upon their binding with folate receptors (Goren et al., 2000), suggesting the potential for such targeting and subsequent internalization strategy in the treatment of several MDR-tumors (Mamot et al., 2003). This approach is additionally supported by the fact that endocytosis of the liposomal drugs is essential for bypassing multidrug resistant (MDR) efflux pumps, such as P-glycoprotein or P-gp, in drug-resistant tumor cells (Gabizon, 2002, Reddy and Low, 1998). Similar results have been obtained with doxorubicin-loaded long-circulating liposomes modified with RGD-peptide motif and capable of targeting the neovasculature of the angiogenic tumors (Xiong et al., 2005a, Xiong et al., 2005b). Using a small cell lung cancer cell line, it was shown that RGD-targeted liposomes were internalized much faster, delivered doxorubicin to the cell nuclei more efficiently, and were more cytotoxic compared to nontargeted liposomes (Moreira et al., 2001). Doxorubicin-loaded liposomes modified with Fab’ fragments of antidisialoganglioside antibodies selectively and almost completely inhibited the metastatic growth of human neuroblastoma in nude mouse model (Pastorino et al., 2003).

From the list of targeting moieties, monoclonal antibodies and their fragments seem to have the highest potential in terms of specificity and variability (Torchilin, 2000). Monoclonal antibodies have been obtained that can recognize specific antigens from the majority of known tumors, such as antibodies against ovarian cancer, prostate cancer, or colorectal cancer (Agus et al., 2000). Earlier, we have identified a family of natural antibodies with nucleosome-restricted specificity, which are capable of effective recognition and binding of a broad variety of live cancer cells (but not normal cells) via the nucleosomes originating from the apoptotically dying neighboring cancer cells and attached to the surface of cancer (but not normal) cells via characteristic nucleosome-binding sites (Iakoubov et al., 1995, Iakoubov and Torchilin, 1998). In addition to their own broad anticancer potential (Chakilam et al., 2004, Torchilin et al., 2003a), these antibodies and their representative, the monoclonal antibody 2C5 (mAb 2C5), being used in subtherapeutic quantities, can serve as effective targeting molecules for tumor-specific delivery of drug-loaded pharmaceutical nanocarriers (Torchilin et al., 2003b). To attach antibodies to Doxil™ liposomes “above” the protective layer of PEG, we have used earlier developed protocol of preliminary antibody modification with p-nitrophenyl-carbonyl-PEG-phosphatidyl ethanolamine (p-NP-PEG-PE) conjugate (Torchilin et al., 2001a) with the subsequent incorporation of the modified antibody molecule into the membrane of PEGylated liposomes via the hydrophobic PE moiety. Earlier, we have obtained some encouraging preliminary data on the increased cytotoxicity of Doxil™ modified by mAb 2C5 (Gupta et al., 2005, Lukyanov et al., 2004).

Here, we present the results of our extended studies on the cytotoxicity of mAb 2C5-modified Doxil™ towards a broad variety of tumor cell lines as well as on the mechanism of the internalization of mAb 2C5-PEG-liposomes by cancer cells.

Section snippets

Materials

Cholesterol (Chol), fully hydrogenated soy phosphatidylcholine (HSPC), N-(carbonyl-methoxy poly (ethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (MPEG2000-DSPE), and phosphatidylehtanolamine (PE) were from Avanti polar lipids, Inc. (Alabaster, AL) and used without further purification. Doxorubicin was purchased from Sigma Chem., Inc. (St. Louis, MO). Triethylamine (TEA), octyl glucoside (OG), and diethylenetriaminepentaacetic acide anhydride (DTPA) were

Results

The method of antibody attachment to liposomes by transferring PEG-PE-modified antibodies from their loose micelles onto the liposome surface used in this study results in attaching approximately 70–80 antibody molecules per single liposome (Gupta et al., 2005, Lukyanov et al., 2004). The antibody incorporation was not accompanied by any significant loss of the liposomal doxorubicin, and no drug release was registered in both the original Doxil™ and the immuno-Doxil™ formulations (modified with

Discussion

The toxic side effects of anticancer drugs, resulting from the lack of specificity of conventional therapies, usually limit the increase in the dose often required to eradicate the cancerous growth. One of the most evolved strategies that have been developed in the last two decades was the enhancement of the tumor-specific delivery of the chemotherapeutic drug, mainly anthracyclins, through actively targeting them via monoclonal antibodies capable of selectively binding antigens that are

Conflict of interest statement

None declared. All authors state that there is no conflict of interest, nor potential conflict of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the work submitted, that could inappropriately influence their work.

Acknowledgements

The authors extend their thanks to Michelle Ocana and Mark Chafel (Harvard Medical School, Center for Neurodegeneration and Repair, Boston, MA) for assisting with the use of their confocal imaging facility. This work was supported by the NIH grant R01 HL55519 to V.P. Torchilin.

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