Saporin is a ribosome-inactivating protein that can cause inhibition of protein synthesis and causes cell death when delivered inside a cell. Development of commercial Saporin results in a technology termed ‘molecular surgery’, with Saporin as the scalpel. Its low toxicity (it has no efficient method of cell entry) and sturdy structure make Saporin a safe and simple molecule for many purposes. The most popular applications use experimental molecules that deliver Saporin via an add-on targeting molecule. These add-ons come in several forms: peptides, protein ligands, antibodies, even DNA fragments that mimic cell-binding ligands. Cells that do not express the targeted cell surface marker will not be affected. This review will highlight some newer efforts and discuss significant and unexpected impacts on science that molecular surgery has yielded over the last almost four decades. There are remarkable changes in fields such as the Neurosciences with models for Alzheimer’s Disease and epilepsy, and game-changing effects in the study of pain and itch. Many other uses are also discussed to record the wide-reaching impact of Saporin in research and drug development.

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Streptavidin-Saporin can be considered a type of ‘secondary’ targeted toxin. The scientific community has taken advantage of this conjugate in clever and fruitful ways using many kinds of biotinylated targeting agents to send saporin into a cell selected for elimination. Saporin is a ribosome-inactivating protein that causes inhibition of protein synthesis and cell death when delivered inside a cell. Streptavidin-Saporin, mixed with biotinylated molecules to cell surface markers, results in powerful conjugates that are used both in vitro and in vivo for behavior and disease research. Streptavidin-Saporin harnesses the ‘Molecular Surgery’ capability of saporin, creating a modular arsenal of targeted toxins used in applications ranging from the screening of potential therapeutics to behavioral studies and animal models. The reagent has become a well-published and validated resource in academia and industry. The ease of use and diverse functionality of Streptavidin-Saporin continues to have a significant impact on the life science industry.

Basigin-2 (EMMPRIN) is a marker for prognosis of several tumor types. With the identification of the internalization of basigin-2 in response to antibody binding, we now further explore the possibility of its use as a dynamic portal of entry for molecules into cancer cells. We also wish to identify a correlation between number of basigin-2 on the cell surface and half maximal effective concentration (EC50) of a targeted toxin by examining eleven tumor cell lines which encompass various tumor types such as breast (MCF-7, BT549), leukemia (K562, HL-60), multiple myeloma (RPMI-8226), neuroblastoma (SH-SY5Y), non-small cell lung (A549, NCI-H23, NCI-H522), and prostate (PC3). In this investigation, we employed the use of flow cytometry to characterize each cell line according to mean fluorescence as well as to determine the number of basigin-2 molecules expressed on the cell surface through a surface quantification assay. This was visualized by staining with a monoclonal antibody directed to basigin-2 ECD with comparison of results against a standard with known Antibody Binding Capacity (ABC) and standardizing the results into Molecules of Equivalent Soluble Fluorochromes (MESF; Bangs Laboratories, Inc.). Last year at this meeting we showed the ability of an antibody to basigin-2 ECD to cause internalization of secondary targeted toxins to cause cell death. We now report on the activity of a primary toxin: the ribosome-inactivating protein saporin attached directly to an antibody to basigin-2. Cytotoxicity assays yielded an EC50 range of 3.5-420 pM while surface quantification assays resulted in expression that ranged from 1-4 million basigin-2 molecules per cell. Cell lines showing potent EC50 values were characterized to have higher surface expression of basign-2 when compared to cell lines with less potent EC50 values. Preliminary results suggest a correlation between EC50 and basigin-2 surface expression such that more basigin-2 available on the cell surface would facilitate greater opportunity for a primary toxin to bind and enter into the cell and cause cell death. These data suggest the use of basigin-2 as a means to identify an entry system for molecules into cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5218. doi:10.1158/1538-7445.AM2011-5218

Basigin-2 (EMMPRIN, CD147) is widely expressed on the surface of numerous tumor types and is associated with poor prognosis. Basigin is an integral membrane glycoprotein also expressed on numerous normal cell types. Several functions have been described for basigin-2 within both normal and malignant tissues. The best characterized is its ability to induce the expression of matrix metalloproteinases in stromal cells. As such, it is suggested to play a role in tumor metastasis. Because of its association with poor prognosis and possible role in metastasis, we examined the ability of basigin-2 to be targeted on the cell surface and internalized as a possible gateway for entry into tumor cells, or for down-regulation for inhibition of metastasis. Belton et al. have documented internalization of basigin-2 by a recombinant soluble protein corresponding to the extracellular domain (ECD) of basigin-2 (J Biol Chem, 2008, 283:17805). We have extended this investigation to examine antibody-mediated endocytosis. A monoclonal antibody to the basigin ECD (also reported in Belton et al.) has been used in an internalization assay using biotinylated antibody mixed with streptavidinylated-saporin (the ribosome-inactivating protein). This complex will eliminate cells only if there is antibody-mediated endocytosis, since saporin has no mechanism of cell entry. HeLa cells, which are known to express basigin-2, were challenged and a potent cytotoxicity was observed: the ED50 of the biotinylated antibody-streptavidin-saporin complex is 370 pM. This is evidence that the antibody internalizes in HeLa cells. Similar data were observed with ovarian carcinoma cells (OVCAR3, SKOV3). Non-tumor cells with low expression have low sensitivity to the complex and there was a correlation between basigin-2 expression on the cell surface and cytotoxicity. We conclude that basigin-2 internalizes in response to antibody binding. Further work is necessary to determine if this can be used for introduction of molecules into basigin-2-expressing cells or if down-regulation would have an effect on metastasis promoted by this poor prognosis molecule. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2244.