Siva AC, Wild MA, Kirkland RE, Nolan MJ, Lin B, Maruyama T, Yantiri-Wernimont F, Frederickson S, Bowdish KS, Xin H (2008) Targeting CUB domain-containing protein 1 with a monoclonal antibody inhibits metastasis in a prostate cancer model. Cancer Res 68:3759-3766. doi: 10.1158/0008-5472.CAN-07-1657
Summary: CUB domain-containing protein 1 (CDCP1) is an antigen expressed on several metastatic cancers, as well as on CD34+ and CD133+ myeloid leukemic blast cells. After demonstrating in vitro activity of the monoclonal antibody 25A11 with Mab-ZAP (Cat. #IT-04) and Hum-ZAP (Cat. #IT-22) the authors had a custom conjugation of 25A11 and saporin made for testing in mice. Goat-IgG-SAP (Cat. #IT-19) was used as a control for in vivo experiments, and saporin (Cat. #PR-01) was the control in vitro. The direct conjugate significantly inhibited tumor growth as well as metastasis in vivo.
One of the tests you can use to test your targeting agent for internalization is the in vitro Cytotoxicity Assay. Protocols to assist in preparing for, executing and interpreting results are now posted on our website.
There are several protocols available.
Preparing for a Cytotoxicity Assay using Secondary Conjugates. This protocol will be helpful when using our secondary antibody-saporin conjugates with your primary antibody. These include Anti-M-ZAP (Cat. #IT-30), Goat-ZAP (Cat. #IT-36), Hum-ZAP (Cat. #IT-22), Mab-ZAP (Cat. #IT-04), Rab-ZAP (Cat. #IT-05), and Rat-ZAP (Cat. #IT-26).
Preparing for a Cytotoxicity Assay using Streptavidin-ZAP. This protocol will be helpful when using our streptavidin-saporin conjugate (Streptavidin-ZAP, Cat. #IT-27) with your biotinylated targeting agent (peptide, ligand, cytokine, growth factor, antibody, etc.).
Concentration Calculation: Convert molarity to mg/ml and mg/ml to molarity. This protocol will help in determining the correct amount of material to use in your assay. There is also a link to an Online Calculator.
Cytotoxicity Assay for Targeted Toxins in vitro. This protocol includes photos of what your plates should look like during the assay process. It takes five days to complete this assay. Start on a Monday and develop on Friday. There are many factors that go into a successful cytotoxicity assay. This protocol should help you design and execute appropriately.
Preparing Cytotoxicity Data. This protocol will give an example of how to process the data from a Cytotoxicity Assay. ATS uses SOFTMax Pro software connected to a plate reader to determine the A490 value. Then we import this data into Prism software (GraphPad) to conduct further data analysis. Here is a figure generated with Prism.
We hope these protocols will be helpful to you in your research. If there are additional protocols or tutorials we can provide, please do not hesitate to ask.
This graph gives important information about how the potency of your targeted toxin. The ED50 is the Median Effective Dose (produces desired effect in 50% percent of population). The lower this number is, the more potent the targeted toxin.
Q: I’m using your secondary conjugate Mab-ZAP (Cat. #IT-04) and it’s not killing my cells. I’m not following the protocol on the data sheet. I’m doing flow cytometry. I have 70,000 cells per well. I mix Mab-ZAP with my primary antibody and add it. When I count the cells, there is no decrease. My cells grow very slowly. I didn’t see anything after 72 hours.
A: The protocol on the data sheet is described in detail in the article by Kohls et al.
70,000 cells per well is a lot of cells per well. We use between 500 and 2500 over a 72-hour period and then develop with MTS.
If your cells are slow-growing, you may want to wait a little longer to develop the assay, because the whole metabolism process is slowed. This is a weakness of the MTS system — you have to have a certain number of cells in the end in the control cells to get a decent reading on your plate reader.
In this case, you might want to try a more sensitive assay such as protein or DNA synthesis inhibition with incorporation of radiolabeled leucine or thymidine.
Q: I purchased your secondary conjugate, Mab-ZAP (Cat. #IT-04). I am preparing to do a cytotoxicity assay and I’m wondering if my primary antibody should be sterilized prior to combining with Mab-ZAP?
A: Depending on the conditions of your lab in which you are using your antibody, it is possible that within a 72-hour period, you may see bacterial growth in your plates if the antibody was accidentally exposed to bacteria. It is recommended that, if you feel comfortable with the antibody, you can just go ahead and try it without sterilizing it, and if you do see bacterial growth, you can certainly filter sterilize the material through a 0.2 micron filter before using.
Bak SP, Walters JJ, Takeya M, Conejo-Garcia JR, Berwin BL (2007) Scavenger receptor-A-targeted leukocyte depletion inhibits peritoneal ovarian tumor progression. Cancer Res 67:4783-4789. doi: 10.1158/0008-5472.CAN-06-4410
Summary: Vascular leukocytes (VLC) are immunosuppressive cells that facilitate tumor progression in ovarian cancer. One potential tumor therapy is to eliminate these cells. The authors determined that scavenger receptor-A is specifically expressed on VLCs. Mice were injected with tumor cells, as well as an anti-scavenger receptor-A antibody combined with Rat-ZAP (Cat. #IT-26). This was followed by additional treatment with the antibody-Rat-ZAP complex. Treatment with the immunotoxin eliminated VLCs, inhibited peritoneal tumor burden, and reduced ascites accumulation.
Elson-Schwab L, Garner OB, Schuksz M, Esko JD, Tor Y (2007) Guanidinylated-Neomycin delivers large, bioactive cargo into cells through a heparan sulfate dependent pathway. J Biol Chem 282(18):13585-13591. doi: 10.1074/jbc.M700463200
Summary: The uptake of high molecular weight drugs into cells is a stumbling block for some potential therapeutics. Using a neomycin derivative in which guanidinium groups have replaced the ammonium groups, the authors show heparan sulfate-dependent uptake of large molecules. The guanidine-neomycin was biotinylated, and incubated with streptavidin-ZAP (Cat #IT-27). This complex was effective in killing CHO cells in vitro, but was no more effective than streptavidin-ZAP alone on cells lacking heparan sulfate expression, demonstrating specificity.
Hess PR, Barnes C, Woolard MD, Johnson MD, Cullen JM, Collins EJ, Frelinger JA (2007) Selective deletion of antigen-specific CD8+ T cells by MHC class I tetramers coupled to the type I ribosome-inactivating protein saporin. Blood 109:3300-3307. doi: 10.1182/blood-2006-06-028001
Objective: To discover if pathogenic T cells could be selectively deleted.
Summary: A single injection of the SAP-coupled tetramer eliminated more than 75% of cognate, but not control, T cells. This work demonstrates the therapeutic potential of cytotoxic tetramers to selectively eradicate pathogenic clonotypes while leaving overall T-cell immunity intact.
Usage: Streptavidin-SAP-coupled biotinylated tetramers were administered at low (22.2 pM) or high (66.6 pM) dose. Following the addition of Saporin Goat Polyclonal, affinity-purified FITC-labeled, T cells were subsequently incubated at either 37°C or 4°C, which permitted or prohibited endocytosis, respectively.
Yip WL, Weyergang A, Berg K, Tonnesen HH, Selbo PK (2007) Targeted delivery and enhanced cytotoxicity of cetuximab-saporin by photochemical internalization in EGFR-positive cancer cells. Mol Pharm 4(2):241-251. doi: 10.1021/mp060105u
Summary: Photochemical internalization (PCI) releases macromolecules from endocytic vesicles using photosensitizer activation by light. This technique allows the release of endocytosed molecules before degradation occurs in the lysosome. The authors demonstrate the proof-of-concept for this technique by combining biotinylated cetuximab (a chimeric monoclonal antibody to the EGFr) with streptavidin-ZAP (Cat. #IT-27). The conjugate was applied to three different human cancer cell lines, demonstrating enhanced specificity and toxicity against cells expressing the EGFr.
Fransson J, Borrebaeck CA (2006) The nuclear DNA repair protein Ku70/80 is a tumor-associated antigen displaying rapid receptor mediated endocytosis. Int J Cancer 119(10):2492-2496. doi: 10.1002/ijc.22212
Summary: The protein Ku70/80 is expressed in the nucleus of all cells. Tumor cell lines, however, have been shown to express Ku70/80 on the cell surface. In this study, the authors show that Ku70/80 is internalized into pancreatic carcinoma cells upon binding of the antibody INCA-X. INCA-X was combined with Mab-ZAP (Cat. #IT-04) and applied to several pancreatic carcinoma cell lines in vitro. Cell death in some of the treated lines demonstrates the potential of Ku70/80 as a therapeutic target.
Collins BE, Blixt O, Han S, Duong B, Li H, Nathan JK, Bovin N, Paulson JC (2006) High-affinity ligand probes of CD22 overcome the threshold set by cis ligands to allow for binding, endocytosis, and killing of B cells. J Immunol 177(5):2994-3003. doi: 10.4049/jimmunol.177.5.2994
Objective: To demonstrate the dynamic equilibrium that exists between CD22 (Siglec-2) and its cis and trans ligands, using a high-affinity multivalent sialoside probe that competes with cis ligands and binds to CD22 on native human and murine B cells.
Summary: The CD22 (Siglec-2) preferred ligand: sequence Siaa2-6Gal that is abundantly expressed on N-linked glycans of B cell glycoproteins. Conjugation of the sialoside probes to the toxin saporin resulted in toxin uptake and toxin-mediated killing of B lymphoma cell lines, suggesting an alternative approach for targeting CD22 for treatment of B cell lymphomas.
Usage: Cytotoxicity assay: BJAB lymphoma cell killing required both the targeting probe and the Streptavidin-ZAP, as no killing was observed in the absence of either.
