zap-conjugates

Foehr ED, Lorente G, Kuo J, Ram R, Nikolich K, Urfer R (2006) Targeting of the receptor protein tyrosine phosphatase beta with a monoclonal antibody delays tumor growth in a glioblastoma model. Cancer Res 66(4):2271-2278. doi: 10.1158/0008-5472.CAN-05-1221

Summary: The receptor protein tyrosine phosphatase ß (RPTPß) is overexpressed in astrocytomas, and is a potential target for tumor therapy. After testing antibodies against an extracellular domain of RPTPß in vitro with Mab-ZAP (Cat. #IT-04), two custom conjugates, 7E4B11-SAP and 7A9B5-SAP, were created by Advanced Targeting Systems. The authors tested the custom conjugates, using anti-DAT-SAP (Cat. #IT-25) as a positive control, and mouse IgG-SAP (Cat. #IT-18) as a negative control. The 7E4B11-SAP conjugate displayed significant antitumor activity in mice engrafted with U87 glioma cells.

Related Products: Mab-ZAP (Cat. #IT-04), Anti-DAT-SAP (Cat. #IT-25), Mouse IgG-SAP (Cat. #IT-18), Custom Conjugates

Q: We are re-examining some data collected using an immunotoxin not prepared by your company (VChAT-sap). Our results in vivo indicated that there were non-specific effects although the creators claimed it was specific. We ran a Western Blot and determined that about half the saporin was not bound to the antibody. This may have been the problem but I want to confirm the cytotoxicity of unbound saporin. Can you confirm that? Further, the antibody in question never bound selectively in ferret tissue. Does this suggest a problem as well? Can you give me information on the best way to design and test an immunotoxin.

A: Yes, saporin at higher concentrations can be cytotoxic. Without specific binding, you will only see non-specific cytotoxicity. The sequence of the immunogen for that antibody is from the rat protein, so I’m not sure if it would target the ferret protein (there usually is good sequence homology among transporters). We worked a lot with this antibody and with an immunotoxin (made by us), but never got any sort of results that would indicate that it was working. We also were greatly concerned that the epitope is an intracellular epitope, and so we have difficulty understanding, from a theoretical standpoint, how it even could work. Because of many concerns, we never commercialized it, and we believe all the effects in the literature were non-specific, but “credible” because of the unusual experimental system that was used.

The best way to design and test an immunotoxin is to talk to us. If your antibody has been tested and shown to be internalized by the cell you are targeting, there should be no problem with the activity. The conjugate will only work as well as your antibody does. We recommend that you try a second immunotoxin before having a custom conjugation performed. This allows you to use a secondary agent conjugated to saporin that “piggybacks” on your antibody and makes a second immunotoxin for use in vitro to test specificity and internalization of your antibody. You can check out the publication that talks about one of the secondary conjugates, Mab-ZAP (Cat. #IT-04). We also have many other secondary conjugates or you can biotinylate your material and use Streptavidin-ZAP (Cat. #IT-27). 

See: ZAP Conjugates, Custom Conjugates

References

1. Kohls MD et al. Mab-ZAP: A tool for evaluating antibody efficacy for use in an immunotoxin. BioTechniques 28(1):162-165, 2000.

Blasius A, Vermi W, Krug A, Facchetti F, Cella M, Colonna M (2004) A cell-surface molecule selectively expressed on murine natural interferon-producing cells that blocks secretion of interferon-alpha. Blood 103(11):4201-4206. doi: 10.1182/blood-2003-09-3108

Objective: To demonstrate the recruitment and function of natural interferon (IFN)-producing cells (IPCs) in murine infection models.

Summary: Incubation of IPCs with the antibody in vitro or administration of the antibody in vivo dramatically reduce secretion of IFN-α in response to deoxycytidylate-phosphatedeoxyguanylate (CpG) DNA without causing IPC depletion. Thus, the antibody identifies an IPC-specific surface molecule that, when engaged, inhibits IFN-α secretion.

Usage: Biotinylated 440c antibody mixed with Streptavidin-ZAP was applied to bone marrow-derived IPCs. Approximately 70% of CD11c+ B220hi 440c bright IPCs were depleted from culture within 36 hours.

Related Products: Streptavidin-ZAP (Cat. #IT-27)

Duxbury MS, Ito H, Ashley SW, Whang EE (2004) CEACAM6 as a novel target for indirect type 1 immunotoxin-based therapy in pancreatic adenocarcinoma. Biochem Biophys Res Commun 317(3):837-843. doi: 10.1016/j.bbrc.2004.03.128

Summary: Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a cell-surface molecule that is overexpressed in a variety of human cancers. Here, the authors investigate the efficacy of a biotinylated antibody that recognizes CEACAM6 bound to streptavidin-ZAP (Cat. #IT-27) in elimination of tumor cells in vitro and in vivo. Treatment of cultured tumor cells induced significant specific cytotoxicity, while tumor growth was suppressed in a mouse xenograft model. These results indicate targeting of CEACAM6 may be a viable therapeutic strategy.

Related Products: Streptavidin-ZAP (Cat. #IT-27)

Q: I recently tried to order avidinylated-SAP (Cat. #IT-09) and was told that this product has been replaced with a new product, Streptavidin-ZAP (Cat. #IT-27). Why did you replace avidinylated-SAP?

A: We initially had good results with avidinylated-SAP. It combined well with biotinylated antibody to produce extremely potent cytotoxic materials, and had low toxicity itself. However, the weaknesses of avidin are well-documented. Probably the most severe is its high isoelectric point that has been suggested to cause nonspecific binding. As we produced more batches of avidinylated-SAP and completed comparative studies, we in fact, found this to be the case.

Q: I use avidinylated-SAP to demonstrate that my antibody internalizes. It worked quite well for me.

A: A couple of months ago we received two reports from customers that they were seeing that, even in batches that had performed well in quality control testing, there was a non-specific cytotoxicity with some cells and/or cell lines. Since a major use of this material is to demonstrate internalization of the biotinylated targeting agent, this was an unacceptable situation. We changed to streptavidin to overcome these specificity issues. As shown in the figure, streptavidin-SAP has an excellent capacity to transform a biotinylated reagent into a potent cytotoxic targeting vehicle, while streptavidin-SAP alone has no detectable cytotoxicity.

See: Streptavidin-ZAP (Cat. #IT-27)

Q: How do I know my antibody will work in an immunotoxin?

A: We recommend that you use one of our second immunotoxins (secondary conjugates) to test for specificity and internalization of your antibody.

Q: How much antibody do I need to provide for a custom conjugation?

A: The required quantity is 8-15 mg of purified monoclonal antibody.

Q: How much of the saporin conjugate will that give me?

A: The yield is 15-30% in mg of immunotoxin from the number of mg of original antibody.

Q: What is the ratio of saporin to antibody?

A: The amount of saporin can vary between 1.5-3 moles saporin per mole of antibody.

Q: What quality control is involved?  Will you provide product specifications such as average saporin to antibody ratio?

A: We monitor the reactions and purification by several means.  The product is confirmed by gel electrophoresis. A data sheet will be provided when we ship the immunotoxin to inform you of final average molecular weight.

Q: What is the cost of a custom immunotoxin preparation? How long will it take to complete?

A: The standard cost of an antibody-saporin conjugation is US$4500.00 (as of Dec 2007).  From the time we receive the purified antibody to the time we ship out the finished conjugate is 2-3 weeks.

See: Custom Conjugates , ZAP Conjugates

Q: What is a second immunotoxin?

A: ATS’s second immunotoxins are conjugations of a secondary antibody (as of December 2000, either goat anti-mouse IgG or goat anti-rabbit IgG) to the ribosome-inactivating protein, saporin.

Q: How does a second immunotoxin target?

A: The second immunotoxin uses the secondary antibody to “piggyback” onto your primary antibody in order to evaluate the ability of the primary antibody to internalize.

Q: What happens when the second immunotoxin gets inside the cell?

A: If the second immunotoxin is internalized, saporin will inactivate the ribosomes of the cell, thereby causing cell death.

Q: Are there different types of second immunotoxins available?

A: Yes, please see our catalog listing online for a complete list.

Q: What is the ratio of antibody to second immunotoxin for in vitro testing?

A: Both Mab-ZAP and Rab-ZAP have been shown effective in concentrations ranging from 0.5 to 2 moles of primary antibody per mole of second immunotoxin.

See: ZAP Conjugates (Second Immunotoxins)

Kohls MD, Lappi DA (2000) Mab-ZAP: A tool for evaluating antibody efficacy for use in an immunotoxin. BioTechniques 28(1):162-165. doi: 10.2144/00281pf01

Summary: Immunotoxins are useful tools for elimination of specific cell populations in vitro and in vivo for research and therapeutic applications. One of the factors limiting the use of immunotoxins is the selection of an appropriate antibody. Advanced Targeting Systems has created a reagent that allows researchers to select antibodies with the desired characteristics before an immuntoxin is made, purified, and assayed. Using a goat anti-murine IgG coupled to the ribosome-inactivating protein saporin, researchers can screen hundreds of antibodies in a time and cost-effective manner.

Related Products: Mab-ZAP (Cat. #IT-04), Rab-ZAP (Cat. #IT-05), Hum-ZAP (Cat. #IT-22), Rat-ZAP (Cat. #IT-26), Anti-M-ZAP (Cat. #IT-30), Goat-ZAP (Cat. #IT-36)

Weltman JK, Melucci CL, Chen J, Davidson AE (1992) Internalization of monoclonal antibodies selected for immunotoxin activity against small-cell lung cancer. Hybridoma 1:547-559. doi: 10.1089/hyb.1992.11.547 PMID: 1334041

Related Products: Mab-ZAP (Cat. #IT-04), Rab-ZAP (Cat. #IT-05), Hum-ZAP (Cat. #IT-22), Rat-ZAP (Cat. #IT-26), Anti-M-ZAP (Cat. #IT-30)

Stirpe F, Barbieri L, Battelli MG, Soria M, Lappi DA (1992) Ribosome-inactivating proteins from plants: present status and future prospects. Bio/Technol 10:405-412. doi: 10.1038/nbt0492-405 PMID: 1368484

Related Products: Saporin (Cat. #PR-01), Saporin Chicken Polyclonal, affinity-purified (Cat. #AB-17AP), Saporin Goat Polyclonal (Cat. #AB-15), MonoBiotin-ZAP (Cat. #BT-ZAP)