title: Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye.
abstract: Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies--Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR)--we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be beneficial for screening a large number of antibody samples during early monoclonal development phase.
authors: Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, United States. Electronic address: nidhi.nath@promega.com.
authors: Nidhi
authors: N
authors: Nath
authors: Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, United States.
authors: Becky
authors: B
authors: Godat
authors: Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, United States.
authors: Chad
authors: C
authors: Zimprich
authors: Promega Biosciences Incorporated, 277 Granada Drive, San Luis Obispo, CA 93401, United States.
authors: Stephen J
authors: SJ
authors: Dwight
authors: Promega Biosciences Incorporated, 277 Granada Drive, San Luis Obispo, CA 93401, United States.
authors: Cesear
authors: C
authors: Corona
authors: Promega Biosciences Incorporated, 277 Granada Drive, San Luis Obispo, CA 93401, United States.
authors: Mark
authors: M
authors: McDougall
authors: Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, United States.
authors: Marjeta
authors: M
authors: Urh
mesh_terms: D000906:Antibodies / Q000032:analysis* / Q000276:immunology; D045744:Cell Line, Tumor; D000068818:Cetuximab / Q000737:chemistry / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D010879:Piperazines / Q000737:chemistry*; D012235:Rhodamines / Q000737:chemistry*; D000068878:Trastuzumab / Q000737:chemistry / Q000276:immunology
chemical_list: D000906:Antibodies; D005456:Fluorescent Dyes; D010879:Piperazines; D012235:Rhodamines; D000068878:Trastuzumab; D000068818:Cetuximab
keywords: Antibody drug conjugate; Antibody internalization; Antibody screening; Confocal microscopy; Homogeneous internalization assay; Plate based internalization assay; pH sensor fluorescent dye