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Targeted delivery of immunotoxin by antibody to ganglioside GD3: A novel drug delivery route for tumor cells.
Torres Demichelis V, Vilcaes AA, Iglesias-Bartolome R, Ruggiero FM, Daniotti JL (2013) Targeted delivery of immunotoxin by antibody to ganglioside GD3: A novel drug delivery route for tumor cells. PLoS One 8(1):e55304. doi: 10.1371/journal.pone.0055304
Summary: The authors used the mouse monoclonal antibody R24 against ganglioside G3 with Mab-ZAP (Cat. #IT-04) to test the viability of ganglioside G3 as a cancer therapy target. Varying concentrations of R24 were used on various cell lines with either 0.95 nM or 9.5 nM Mab-ZAP depending on the cell line.
Related Products: Mab-ZAP (Cat. #IT-04)
CD22 antigen is broadly expressed on lung cancer cells and is a target for antibody-based therapy.
Tuscano JM, Kato J, Pearson D, Xiong C, Newell L, Ma Y, Gandara DR, O’Donnell RT (2012) CD22 antigen is broadly expressed on lung cancer cells and is a target for antibody-based therapy. Cancer Res 72(21):5556-5565. doi: 10.1158/0008-5472.CAN-12-0173
Summary: The median overall survival of patients with advanced, unresectable, non-small cell lung cancer is 9-12 mos. A potential therapeutic target is CD22, a protein expressed on lung cancer cells. The authors examined the use of the monoclonal antibody HB22.7 as an antitumor agent. To assess internalization of the antibody, it was first incubated with 10 μg/ml Mab-ZAP (Cat. #IT-04) then applied to two different cancer cell lines in culture. Analysis of cell viability demonstrated that CD22 internalized when bound by the antibody-toxin complex, suggesting that targeting CD22 has therapeutic potential.
Related Products: Mab-ZAP (Cat. #IT-04)
Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1.
Daniels-Wells TR, Helguera G, Rodriguez JA, Leoh LS, Erb MA, Diamante G, Casero D, Pellegrini M, Martinez-Maza O, Penichet ML (2013) Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1. Toxicol In Vitro 27(1):220-231. doi: 10.1016/j.tiv.2012.10.006
Summary: The antibody-avidin fusion protein ch128.1Av has been shown to target the human transferrin receptor 1 (TfR1) and kill malignant B cells by blocking the use of iron. Combination of this construct with a mono-biotinylated saporin custom conjugate produces an iron-independent toxicity to TfR1-expressing cells, even those that are resistant to ch128.1Av alone. The saporin-containing conjugate induces a transcriptional response consistent with oxidative stress and DNA damage. The data also show that the saporin conjugate is not toxic to human hematopoeietic stem cells.
Usage: An antibody-avidin fusion protein (ch128.1Av) was mixed with MonoBiotin-ZAP to make an immunotoxin that targets the human transferrin receptor 1 (TfR1).
Related Products: MonoBiotin-ZAP (Cat. #BT-ZAP), Custom Conjugates
Photochemical internalization (PCI) of HER2-targeted toxins: Synergy is dependent on the treatment sequence.
Berstad MB, Weyergang A, Berg K (2012) Photochemical internalization (PCI) of HER2-targeted toxins: Synergy is dependent on the treatment sequence. Biochim Biophys Acta 1820(12):1849-1858. doi: 10.1016/j.bbagen.2012.08.027
Summary: A majority of patients develop acquired resistance to trastuzumab, the monoclonal antibody recognizing HER2, coupled to a toxin as a breast cancer therapeutic. One of the modes of resistance is that the therapeutic molecule is trapped inside an endocytic vesicle. PCI is a technique that facilitates cytosolic release of molecules in vesicles. The authors investigated the potency of biotinylated trastuzumab combined with streptavidin-ZAP (Cat. #IT-27) on several cell lines.
Related Products: Streptavidin-ZAP (Cat. #IT-27)
Identification of a cKit(+) colonic crypt base secretory cell that supports Lgr5(+) stem cells in mice
Rothenberg ME, Nusse Y, Kalisky T, Lee JJ, Dalerba P, Scheeren F, Lobo N, Kulkarni S, Sim S, Qian D, Beachy PA, Pasricha PJ, Quake SR, Clarke MF (2012) Identification of a cKit(+) colonic crypt base secretory cell that supports Lgr5(+) stem cells in mice. Gastroenterology 142:1195-1205.e1196. doi: 10.1053/j.gastro.2012.02.006
Objective: To investigate the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5 stem cells.
Summary: cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5+ stem cells.
Usage: For saporin experiments, small intestinal organoids with crypt buds and visible Paneth cells were passaged as single cells. Before embedding in Matrigel, dissociated cells were incubated with Anti-cKit-SAP (biotinylated-2B8 mixed with Streptavidin-ZAP). Treatment led to a marked decrease in organoid formation.
Related Products: Streptavidin-ZAP (Cat. #IT-27)
Strongly amphiphilic photosensitizers are not substrates of the cancer stem cell marker ABCG2 and provides specific and efficient light-triggered drug delivery of an EGFR-targeted cytotoxic drug.
Selbo PK, Weyergang A, Eng MS, Bostad M, Maelandsmo GM, Hogset A, Berg K (2012) Strongly amphiphilic photosensitizers are not substrates of the cancer stem cell marker ABCG2 and provides specific and efficient light-triggered drug delivery of an EGFR-targeted cytotoxic drug. J Control Release 159(2):197-203. doi: 10.1016/j.jconrel.2012.02.003
Summary: Many anti-cancer drugs are substrates of the ATP-binding cassette transporter ABCG2. Unfortunately ABCG2 is also thought to play an important role in multi-drug resistance and the protection of cancer stem cells against chemotherapeutics and photodynamic therapy. This paper examined whether photosensitizers used in photochemical internalization (PCI) are substrates for ABCG2. Streptavidin-ZAP (Cat. #IT-27) was combined with biotinylated EGF and applied to cells in culture; saporin (Cat. #PR-01) was used as a control. The data show that PCI with the EGF-saporin toxin did not utilize ABCG2 to enter cells.
Related Products: Streptavidin-ZAP (Cat. #IT-27), Saporin (Cat. #PR-01)
Selective targeting of microglia by quantum dots.
Minami SS, Sun B, Popat K, Kauppinen T, Pleiss M, Zhou Y, Ward ME, Floreanig P, Mucke L, Desai T, Gan L ( 2012 ) Selective targeting of microglia by quantum dots. J Neuroinflammation 9(1):22 . doi: 10.1186/1742-2094-9-22
Related Products: MonoBiotin-ZAP (Cat. #BT-ZAP)
Choosing the Correct Secondary Conjugate
Q: I have a mouse monoclonal antibody and a rabbit polyclonal antibody that I would like to test using your secondary conjugate system. Which products do I need to order?
A: For mouse monoclonals, you can use: Mab-ZAP (Cat. #IT-04) – Cells that internalize your mouse monoclonal antibody will be eliminated. Or, Fab-ZAP mouse (Cat. #IT-48) – Cells that internalize your mouse monoclonal IgG antibody will be eliminated.
For rabbit polyclonals, you can use: Rab-ZAP (Cat. #IT-05) – Cells that internalize your rabbit polyclonal antibody will be eliminated. Or, Fab-ZAP rabbit (Cat. #IT-57) – Cells that internalize your rabbit IgG antibody will be eliminated.
The difference between Fab-ZAP products and other secondary conjugates is that Fab-ZAP is made with a monovalent secondary antibody which eliminates the possibility of cap formation as cross-linking of the Fab-ZAP molecules cannot occur. The Fab-ZAP products will still recognize the heavy and light chains of antibodies, and should be used in the same way and at the same molar concentrations as the original secondary conjugates (such as Mab-ZAP and Rab-ZAP). Cytotoxicity assays using Fab-ZAP (mouse) have demonstrated an improved EC50 when directly compared to Mab-ZAP.
Any of our secondary conjugates offer a very cost-effective diagnostic method for screening primary antibodies for in vitro or in vivo use.
Related Products: ZAP Conjugates
Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and Treg cells
Facciabene A, Peng X, Hagemann IS, Balint K, Barchetti A, Wang L-P, Gimotty PA, Gilks CB, Lal P, Zhang L, Coukos G (2011) Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and Treg cells. Nature 475:226-230. doi: 10.1038/nature10169 PMID: 21753853
Objective: To investigate whether a direct link between tumor hypoxia and tolerance occurs through the recruitment of regulatory cells.
Summary: Tumor hypoxia promotes the recruitment of regulatory T (Treg) cells through induction of expression of the chemokine CC-chemokine ligand 28 (CCL28), which, in turn, promotes tumor tolerance and angiogenesis.
Usage: In vivo depletion of CD4+ CD25+ cells was achieved by intraperitoneal administration of anti-CD25 or an immunotoxin consisting of anti-mouse CCR10 or anti-mouse CCR3 antibody conjugated at an equimolar ratio to Streptavidin–ZAP. Anti-CCR10–SAP depleted 90% of CCR101 or CCR31 cells. Anti-CCR10–SAP suppressed tumour growth and abrogated the effects of CCL28 overexpression, whereas anti-CCR3–SAP had no effect on tumor growth.
Related Products: Streptavidin-ZAP (Cat. #IT-27)
Higher concentration and less cytotoxicity
Q: We’ve been using Mab-ZAP to test our primary mouse monoclonal antibody in cytotoxicity assay. Relative to the Primary mAb-Mab-ZAP complex, the Mab-ZAP alone has quite a bit of activity (40-60% growth inhibition) at the recommended concentrations used (45 or 100 ng/well) or even half that dose. Even though we get a dose response, the higher concentrations of primary antibody give me less cytotoxic activity than the lower concentrations. Can you give your thoughts on this matter?
A: The effect you are seeing is something that is actually typical and indicates that you are using the material correctly. Described in Kohls et al., unbound primary antibody will compete with primary antibody bound to a secondary conjugate may reduce cytotoxicity through competitive inhibition of the primary antibody-Mab-ZAP complex. This is especially noticeable with our Fab-ZAP line of secondary conjugates.
We still recommend that our customers try a 10 nM dose as a starting point, but always recommend adjusting the concentration to better accommodate their experiments. As a reference, our data sheets show a cytotoxicity curve with a starting concentration of 10 nM and an ending concentration of 1 fM.
Related Product: Mab-ZAP (Cat. #IT-04)
References