Contributed by Jose Luis Daniotti, Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC, UNC-CONICET),
Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.
Glycosphingolipids (GSLs) are amphipathic molecules consisting of a ceramide lipid moiety linked to a glycan chain of variable length and structure. Among these are found the gangliosides, which are mono- or multi-sialosylated GSLs mainly located in the outer layer of the plasma membrane of vertebrate cells. These are expressed in cell type- and developmental-specific patterns and are major components of nerve cells, being implicated in many physiological processes, including growth, differentiation, migration and apoptosis. Furthermore, gangliosides have been associated with a wide range of pathological processes, as they are receptors for both viruses and antibodies. 
The biosynthesis of gangliosides starts at the endoplasmic reticulum and continues through a combination of glycosyltransferase activities at the Golgi complex, which is followed by vesicular delivery to the plasma membrane. [1-3] Recently, our laboratory and other researchers have demonstrated that the regulation of GSL expression also occurs at the cell surface, where both glycosyltransferases and glycohydrolases locally modulate the cellular glycolipid pattern. [4-6]
Over recent years, we have paid particular attention to the synthesis and function of ganglioside GD3. Although this disialo glycolipid is highly expressed at the early developmental stages of the central nerve system, at later developmental stages the GD3 content declines and other gangliosides become major players. [1,3] Despite the expression levels of gangliosides in general, and of GD3 in particular, being very low and restricted to adult extra neural tissues, GD3 is highly expressed in tumor cells, especially melanomas. It is also overexpressed in neuroectodermal tumors (neuroblastoma and glioma) and carcinomas of lung, breast, colon, prostate and ovary.  For these reasons, ganglioside GD3 has received considerable attention as a promising immunotherapeutic target for cancer therapy. As such, it has been used for passive and active immunotherapy of melanoma cancer, but with antibody therapy only producing modest results.
Mouse monoclonal R24 antibody (IgG3), directed against ganglioside GD3, is a validated tumor targeting agent that shows strong cell surface reactivity for a range of human melanoma cell lines and other epithelial cancer tumor cells.  We demonstrated that in different cell lines the R24 antibody to GD3 is rapidly endocytosed after binding to the disialo ganglioside at the cell surface, sorted to early endosomes and later accumulated in recycling endosomes (Fig. 1).  Consequently, its rapid internalization in cells precludes its use as a “naked therapeutic antibody,” because when internalized it cannot link to pathways of complement- or cellular- dependent anticancer activities.
We took advantage of the internalization feature of R24 antibody for selective delivery of saporin to GD3-expressing cells.  This was carried out by mixing the R24 antibody with Mab-ZAP (Cat. #IT-04), a goat anti-mouse IgG antibody linked to saporin. The targeted toxin was found to be specifically cytotoxic for GD3-expressing cells [human (SK-Mel-28), mouse (B16) melanoma cells and CHO-K1 cells] grown on 2D monolayers. To estimate the potential antitumor activity of the R24/Mab-ZAP complex, we also evaluated the effect of the targeted toxin on clonogenic growth of cells in attachment-free conditions. Briefly, cells expressing the ganglioside GD3 were grown in semi-solid medium for seven days to allow the formation of colonies containing approximately 60-80 cells. Then, cells were exposed to the R24/Mab-ZAP complex and the size of the colony was scored at different times. Surprisingly, a drastic growth inhibition of SK-Mel-28 melanoma cells was reached after only 3 days of treatment (Fig. 2). In contrast, cell colonies continued to grow with the same concentration of the targeted toxin, but in the absence of the R24 antibody, or in the absence of both targeted toxin and R24, undoubtedly indicating the specificity of the effect observed.
Antibody-drug conjugates are emerging as highly effective therapies for cancer. From our studies, the ganglioside GD3 has emerged as a novel and attractive class of cell surface molecule (glycosphingolipid) for targeted delivery of drugs. This is due to its accessibility, low expression in normal cells, high expression in many tumor cells (mainly of neuroectodermal and epithelial origin) and for its capacity to undergo endocytosis after binding with extracellular ligands such as antibodies. We believe that it is possible to potentiate the cytotoxic properties of R24 on target cells by linking it to other drugs such as paclitaxel, which will aid in bypassing multiple drug resistance mediated by the p-glycoprotein pumps.
Internalization of antibody to GD3 (R24) in human SK-Mel-28 melanoma cells. Human SK-Mel-28 cells, previously characterized as expressing endogenous GD3, were incubated with antibody to GD3 for 45 min at 4ºC (top panel). Then, the temperature was shifted to 37 ºC to allow endocytosis of the complex GD3-R24 and cells were fixed at 30 min (bottom panel). R24 antibody was detected by using anti-mouse IgG conjugated with Alexa488. Single confocal sections of 0.8 µm were taken parallel to the coverslip.
Modified from Ref. 9.
Selective delivery of saporin via R24 antibody drastically reduces the clonogenic growth of human SK-Mel-28 melanoma cells.
A) A schematical representation of the experimental procedure used in B.
B) SK-Mel-28 cells (50-80 cells) were grown in 24-well plates previously coated with 0.5% agar in culture medium. Cells were maintained at 37ºC until cell colonies appeared (7 days). Then, cells were exposed for 3 days (7+3 days) to 0.95 nM Mab-ZAP (Saporin-Ab) or 30 nM R24/0.95 nM Mab-ZAP (R24/Saporin-Ab). Quantification was performed at 7 and 7+3 days. SK-Mel-28 cells maintained only with medium were used as negative control (control). Results were analyzed by ANOVA followed by Tukey’s multiple comparison test. Results are given as means ±S.E. Note that the clonogenic growth of SK-Mel-28 cells was severely affected only in the presence of the R24/Saporin-antibody complex
(***p<0.0001 with respect to the control condition at 7+3 days).
Modified from Ref. 10.
References: (back to top)
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