A Fluorescence-Based Method to Measure Antibody Internalization in Tumor Cells Haibiao Gong, Teresa Urlacher LI-COR Biosciences, Lincoln, NE 68504 A BSTRACT The outcome of many antibody-based therapies, such as antibody-drug conjugates, immunotoxins, liposomal drugs, and targeted gene delivery, depends not only on the affinities and specificities of the antibodies, but also on their internalization after antigen binding. Various methods have been developed to analyze antibody internalization. The traditional method relies on an acidic buffer to dissociate the surface-bound radioisotopelabeled antibody. A variation of this method utilizes a fluorophore for signal detection and an anti-fluorophore antibody to quench the surfaceassociated fluorescence. Antibodies attached with an immunotoxin or drug that can kill cells after internalization have also been employed as reporters of antibody internalization. In this report, we developed a simple, sensitive, fluorescence-based method to monitor antibody internalization. Panitumumab, an anti-EGFR therapeutic antibody, was labeled with the fluorescent dye IRDye® 800CW (LI-COR Biosciences) and quencher M ATERIALS AND IRDye® QC-1 (LI-COR Biosciences), and designated as Pan800QC. The fluorescence of 800CW is quenched by QC-1 labeled on the same antibody molecule. After incubation with EGFR-expressing cells, internalization of Pan800QC was detected by the increase of fluorescence signal due to enzymatic cleavage of the antibody and separation of 800CW and QC-1. By optimizing the reaction conditions, including cell density, Pan800QC concentration, and incubation time, a signal-to-background ratio of 8.5 was obtained. This homogeneous assay can be applied in the analysis and screening of internalizing antibodies. I NTRODUCTION Upon target binding, antibodies are often internalized into the cells and digested by the proteases in lysosomes. Internalization is a prerequisite for most antibody-based drug delivery systems. Internalization may also influence the quality of molecular imaging with targeting probes. Therefore, analysis of antibody internalization after target binding is essential for these applications. In this study, we developed a simple, but robust fluorescence-based method for antibody internalization assay. In this method, the antibody is labeled with a fluorescent dye (IRDye 800CW) and a quencher (IRDye QC-1). The fluorescence of 800CW is quenched by QC-1 labeled on the same antibody molecule. The fluorescence will be restored after antibody internalization and degradation. Using this strategy, panitumumab, an anti-EGFR therapeutic antibody, was labeled and tested. After incubation with EGFR-expressing F98-EGFR cells, the labeled probe (named as Pan800QC) was internalized by the cells upon EGFR binding. The fluorescence signal, resulting from the antibody degradation and separation of IRDye 800CW from QC-1, was detected using an Odyssey® CLx Imager (LI-COR Biosciences). The fluorescence signal intensity indicates the amount of internalized antibody and the extent of degradation. A more general method employing a secondary antibody or protein A/G that is able to associate with target-binding primary antibodies could be used for internalizing antibody screening. M ETHODS Figure 1. Panitumumab labeled with IRDye 800CW (D/P=1.5) and different numbers of IRDye QC-1 quencher. (A) Gel analysis of labeled probes. (B) Quenching efficiency of labeled probes. Note that QC3, QC6, QC9, and QC12 designate the number of QC-1 molecules per antibody in the labeling reaction (3, 6, 9 and 12, respectively). The actual numbers of QC-1 on the antibody are unknown. QC0 designates panitumumab labeled with IRDye 800CW without QC-1. QC9 probe (named as Pan800QC) was used for subsequent studies. Figure 4. The signal-to-background ratio (SBR) was dependent on Pan800QC probe concentration and cell density. (A) Different concentrations (0.016 to 16 nM) of Pan800QC were incubated with F98-p or F98-EGFR cells for 24 h. (B) Pan800QC (0.5 nM) was incubated with different numbers of F98-p or F98EGFR cells for 24 h. SBR was calculated by dividing the F98-EGFR signal by F98-p signal. Figure 2. Gel analysis of internalized probes. Quenched probe Pan800QC (lanes 2 and 3) and unquenched probe Pan800 (lanes 5 and 6) were incubated with cells without human EGFR (F98-p, lanes 2 and 5) or cells expressing EGFR (F98-EGFR, lanes 3 and 6). After 24 h of incubation, cells were lysed and analyzed by gel electrophoresis. Probes Pan800QC and Pan800 (lanes 1 and 4) and IRDye 800CW carboxylate dye (lane 7) diluted in PBS were loaded on the gel as controls. Both Pan800QC and Pan800 were internalized and degraded in F98-EGFR cells. Note that the signal of intact Pan800QC was much lower than that of Pan800. Figure 5. Pan800QC internalization was blocked by EGFR binders. (A) Pan800QC (0.5 nM) was incubated with F98-EGFR in the presence of 50 nM of different competitors. Pan: panitumumab; Cet: cetuximab; 7D12: EGFR-specific nanobody; EGF: epidermal growth factor; E-aff: EGFR-specific affibody; H-aff: HER2-specific affibody. (B) Pan800QC (0.5 nM) was incubated with F98-EGFR in the presence of different concentrations (0.5 to 500 nM) of cetuximab or panitumumab. Signals were measured after 24 h of incubation. Figure 3. Fluorescence signals and signal-tobackground ratio (SBR) of Pan800QC after EGFR-meditated internalization. (A) Pan800QC was incubated with F98-p (non-EGFR-expressing) or F98-EGFR (EGFRexpressing) cells for different time periods. Fluorescence signals were detected on the Odyssey CLx Imager. (B) SBR was calculated by dividing the F98EGFR signal by F98-p signal. The SBR was highest after 24 h of incubation. C ONCLUSION A homogeneous, fluorescence-based method to monitor antibody internalization was established and tested using panitumumab as an example. The assay was performed in 96-well plates with a simple procedure in which the probe was added to the cell culture medium directly. After incubation, the fluorescence signal was detected in the plate. By optimizing the reaction conditions, including cell density, the Pan800QC concentration, and incubation time, a signal-to-background ratio of 8.5 was obtained. This method can be used for internalizing antibody screening and analysis. LI-COR is an ISO 9001 registered company. © 2014, LI-COR, Inc. LI-COR, IRDye, and Odyssey are trademarks or registered trademarks of LI-COR, Inc. All other trademarks belong to their respective owners. For patent information, visit www.licor.com/patents. Presented at Keystone Symposia on Molecular and Cellular Biology, February 2014. www.licor.com