pHast-conjugates

Question:

We have been using your human fab-pHast (PH-01) product for a while now and are very happy with the results in our imaging applications (we have seen signal to noise of over 70k). However I have recently tried running a black bottom plate on a plate reader, same set up, and am getting a high background signal. My signal to noise is about 1.5 in the best case scenario. Could you please advise how to increase the signal to noise in the plate reader format using fab-pHast?

Answer:

We recommend washing the cells with PBS before reading on the plate reader to help reduce background signal. You can find an internalization assay protocol here.

Question:

I’m using complete media (phenol red DMEM with 10% FBS) for my experiments, could this be contributing to background signal?

Answer:

Using phenol red-containing media could contribute to background signal. Replacing the media with PBS should reduce background significantly.

Question:

Do you have any recommendations on whether to use black opaque-bottom plates, black clear-bottom plates, or standard clear TC plates for maximal S/N?

Answer:

Clear-bottom plates offer the advantage of imaging, but they may introduce more background noise. If imaging is a priority, you might need to balance between signal strength and versatility.

Related Products

pHast Conjugates:

View a table of targets and recommended pHast conjugates for testing your primary antibody specificity.

Shramm PA, Ancheta L (2019) Streptavidin-pHast: A readily conjugatable, pH-sensitive dye to screen for internalization. Cancer Res 79(13 Suppl):2177. Proceedings of the American Association for Cancer Research Annual Meeting, Atlanta GA. PMID: 909090

Summary: Quick and efficient screening of targeting agents that internalize effectively is vital for determining their suitability as potential therapeutics. Some of the most recent successes in the treatment of cancers have been from antibodies to cell surface proteins that are responsible for tumor cell proliferation. Examples are Cetuximab (target: EGFR) approved for colorectal cancer, and Trastuzumab (target: HER2) for breast cancer. These antibodies have more than one effect on the cancer cell, but one of the most important is that, upon binding to the cell surface antigen, the complex is internalized. As such, the down-regulated cell surface protein no longer plays a role in cancer cell division. Here we describe a method for determining internalization of cell surface molecules by targeting agents using a pH-dependent fluorescent reporter cross-linked to streptavidin. Streptavidin is a tetrameric protein (molecular weight 53 kDa in its recombinant form), with each subunit able to bind a single biotin molecule. The bond between streptavidin and biotin is rapid and essentially non-reversible, unaffected by most extremes of pH, organic solvents, and denaturing reagents. It is the strongest known noncovalent biological interaction (Ka = 1015 M-1) between protein and ligand. A variety of molecules, including lectins, proteins, and antibodies, can be biotinylated and reacted with streptavidin-labeled probes or other detection reagents for use in biological assays. The fluorescence from this reporter increases intensity as the pH of its surroundings becomes more acidic, as demonstrated when exposed to the environment inside a cell (thereby providing evidence of internalization). Here we describe methods that can be used to explore candidates as cancer therapeutics in a quick, reliable and reproducible manner.

Related Products: Streptavidin-pHast (Cat. #PH-03)

View complete Poster.

See Also:

• Zangardi ML et al. Sacituzumab for the treatment of triple-negative breast cancer: the poster child of future therapy?. Expert Opin Investig Drugs 28(2):107-112, 2019.
• Lamb YN Inotuzumab ozogamicin: First global approval. Drugs 77(14):1603-1610, 2017.
• Kinneer K et al. SLC46A3 as a potential predictive biomarker for antibody-drug conjugates bearing noncleavable linked maytansinoid and pyrrolobenzodiazepine warheads. Clin Cancer Res 24(24):6570-6582, 2018.
• Facciabene A et al. Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and Treg cells. Nature 475:226-230, 2011.
• Kohls M Evaluate Potential Targeting Molecules. Nature Methods , 2006.

Kinneer K, Meekin J, Tiberghien AC, Tai YT, Phipps S, Kiefer CM, Rebelatto MC, Dimasi N, Moriarty A, Papadopoulos KP, Sridhar S, Gregson SJ, Wick MJ, Masterson L, Anderson KC, Herbst R, Howard PW, Tice DA (2018) SLC46A3 as a potential predictive biomarker for antibody-drug conjugates bearing noncleavable linked maytansinoid and pyrrolobenzodiazepine warheads. Clin Cancer Res 24(24):6570-6582. doi: 10.1158/1078-0432.CCR-18-1300 PMID: 30131388

Objective: To develop biomarkers to uncover the underlying mechanism of resistance by certain cell lines for ADCs.

Summary: Loss of SLC46A3 expression was found to be a mechanism of innate and acquired resistance to ADCs bearing DM1 and SG3376.

Usage: For Lysosomal trafficking, ADCs were labeled with Fab-pHast human (Cat. #PH-01). Cells were incubated with 3 mg/mL of labeled ADCs at 37°C for desired time points and fluorescence quantified by flow cytometry.

Related Products: Fab-pHast human (Cat. #PH-01)

Shramm PA, Ancheta L, Higgins D, Lappi DA (2017) A rapid, pH-sensitive screening method to detect internalization of cell surface markers for development of antibody-based pharmaceuticals to treat brain tumors. Neuroscience 2017 Abstracts 566.24 / H7. Society for Neuroscience, Washington, DC.

Summary: Some of the most potent treatments for cancers have been antibodies to cell surface proteins that cause tumor cell proliferation. Examples are cetuximab (antigen: EGFR) approved for colorectal cancer and Trastuzumab (ERBB2) for breast cancer. These antibodies have more than one effect on the cancer cell, but one of the most important is that, upon binding to the cell surface antigen, the complex is internalized by so-called antibody mediated internalization. As such, the mitogenic cell surface protein no longer plays a role in cancer cell division. Despite the blood brain barrier challenging systemic treatment for brain tumors, intracerebroventricular injection can produce similar results. For example, Gholamin et al., (Sci Transl Med 9:381, 2017) and Kang et al. (Sci Rep 6:34922, 2016) reported down-regulation of brain tumor mitogenic agents through antibody-mediated endocytosis. The quick and efficient screening of antibodies that internalize effectively is vital for determining suitability of an antibody as a therapeutic targeting agent. Here we describe a method for the efficient determination of internalization of cell surface molecules by antibodies using a pH-dependent fluorescent reporter cross-linked to a secondary antibody in a plate-based assay with visualization of internalization in hours. This conjugate is comprised of an affinity-purified monovalent secondary antibody against both the heavy and light chain of human or mouse IgG and is conjugated to a pH -dependent fluorescent reporter. The fluorescence from this reporter increases intensity as the pH of its surroundings becomes more acidic, as evident when exposed to the environment inside a cell (thereby providing evidence of internalization). A successful assay protocol has been developed to provide an EC50 by way of a fluorescence-detecting plate reader, which could be used to explore antibody candidates as therapeutics in a quick and reproducible manner.

Related Products: Fab-pHast human (Cat. #PH-01)

View complete Poster.

See Also:

• Gholamin S et al. Disrupting the CD47-SIRPα anti-phagocytic axis by a humanized anti-CD47 antibody is an efficacious treatment for malignant pediatric brain tumors. Sci Transl Med 9(381):eaaf2968, 2017.
• Kang BR et al. Cell surface GRP78 as a biomarker and target for suppressing glioma cells. Sci Rep 6:34922, 2016.
• Ancheta LR et al. Basigin-2 (EMMPRIN), a prognostic marker, is a dynamic portal of entry into cancer cells. Cancer Res 71(8):5218, 2011. Proceedings of the American Association for Cancer Research Annual Meeting, Orlando, FL
• Ancheta L et al. Method for screening neuronal tumor cell surface markers for high specificity and rapid internalization as potential oncologic treatments. Neuroscience 2017 Abstracts 612.11 / SS46, 2017. Society for Neuroscience, Washington, DC

Ancheta L, Shramm PA, Lappi DA (2017) Method for screening neuronal tumor cell surface markers for high specificity and rapid internalization as potential oncologic treatments. Neuroscience 2017 Abstracts 612.11 / SS46. Society for Neuroscience, Washington, DC.

Summary: Targeted cancer therapies are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules involved in the growth, progression, and spread of the tumor. These therapies are often cytostatic; they block tumor cell proliferation as opposed to chemotherapy that kills the cells. A primary approach to identify potential targets is the ability to compromise a ligand/receptor relationship that causes tumor cell proliferation. There are now many examples of the use of antibodies in tumor therapy to cause a breakdown in that relationship. In clinical use against brain tumors are antibodies to cell-surface EGFR, VEGFR, PDGFR, and c-kit. These work by down-regulation of the receptor by antibody-mediated internalization. It is crucial for development of a targeted therapy to have a method to determine the suitability of an antibody to cause internalization rapidly and completely. Here we describe a method for the efficient determination of internalization of cell surface molecules by antibodies: a cytotoxicity assay utilizing an antibody labeling method to streamline the process of multiple candidate screening. Cells are chosen that have significant levels of expression of the desired marker and the assay readout is definitive: cell death is demonstrated in 72 hours. This method is designed for the rapid screening of multiple antibodies for specificity and internalization in neuronal tumor cells to explore antibody candidates as therapeutics in a quick and reproducible manner.

Related Products: Fab-pHast human (Cat. #PH-01)