Tools for studying antibody internalization and trafficking

(See a list of the products featured in this article.)

Therapeutic monoclonal antibodies (mAbs) and their derivatives represent an exciting and rapidly developing class of medicines that are used to treat serious diseases such as cancer, autoimmunity, and metabolic disorders [1]. These agents include antibody–drug conjugates (ADCs) and bi-specific variants, which are engineered to recognize two different antigens. Produced using recombinant DNA technology, therapeutic mAbs bind specific soluble or membrane-bound target molecules to alter cell signaling events. Because they closely resemble the human body’s own immunoglobulin (Ig) molecules, therapeutic mAbs are generally well tolerated and have favorable safety profiles. Several different mechanisms of action for mAb-based molecules are shown in Figure 1.

With the intensifying interest in mAb technologies from the biopharmaceutical sector as well as academic investigators, there is a growing need for improving and expanding the range of tools for studying therapeutic mAbs as they engage with their cellular and subcellular targets. In the case of an ADC, for example, the internalization and trafficking of the conjugate to specific cellular compartments are fundamental to the drug’s mechanism of action [2]. Investigators may need to assess the propensity of a therapeutic mAb candidate for degradation versus recycling pathways as a component of pharmacokinetic analysis [3]. In addition, preclinical safety and efficacy studies may require whole-animal noninvasive imaging studies to track the biodistribution of a drug or surrogate in tissues [4]. Scientists at Thermo Fisher Scientific have developed a wide range of solutions to address these and other important questions for investigators engaged in the discovery and advancement of therapeutic mAbs.

Figure 1. Therapeutic mAbs and their derivatives can affect target cell function and viability via several different mechanisms. For oncology indications where directed tumor cell lysis is the desired endpoint, mAbs can be used to engage cytotoxic or phagocytic effector cells (ADCC or ADCP, respectively), to drive pro-apoptotic signaling via ligation of death receptors (PCD), or to activate complement cascades (CDC). An antibody–drug conjugate (ADC) consists of a mAb directed against a tumor cell antigen coupled to a small cytotoxic molecule, resulting in a highly specific and targeted chemotherapeutic agent.  

Selected technologies for fluorescent antibody labeling

During target validation and selection of lead candidates, it is often advantageous to rapidly and reliably label mAbs with fluorescent dyes to facilitate their detection in imaging or flow cytometry studies. We offer a variety of conjugation technologies designed to help researchers generate fluorescent antibodies custom-labeled with Molecular Probes™ dyes (Table 1).

APEX™ Antibody Labeling Kits are the preferred method when the goal is to quickly label a small quantity (10–20 μg) of antibody that is suspended in a buffer containing other proteins, such as albumin or gelatin, which cannot easily be dialyzed away. These kits utilize a solid-phase labeling technique that captures the IgG antibody on a resin inside an APEX antibody labeling tip. By immobilizing the IgG within a micro-column, stabilizing proteins are easily eluted prior to the solid-phase conjugation step, which uses an amine-reactive fluorophore that reacts with the antibody’s lysines. The fluorescent IgG conjugate is ready to use in an imaging or flow cytometry assay in as little as 2.5 hours, with minimal hands-on time.

Labeling antibodies for use as imaging reagents in whole-animal biodistribution studies presents unique challenges. Importantly, the degree of labeling (DOL), which refers to the number of dye molecules per antibody, must be tightly controlled to maximally preserve the antibody’s binding characteristics. Therefore, the dyes themselves must have high quantum yield for accurate and sensitive detection in the near-infrared spectrum. Also, these experiments typically require larger amounts of antibody than many labeling kits can accommodate. The SAIVI Rapid Antibody Labeling Kits were specifically designed to address these needs, allowing researchers to conjugate their own primary antibodies of choice at milligram scale to the extremely bright and photostable near-infrared–fluorescent Alexa Fluor™ 680 and Alexa Fluor 750 dyes with a high degree of precision and confidence. Fluorescent antibody conjugates are eluted in sterile azide-free buffer and do not require additional dialysis steps prior to in vivo administration.

A different approach to fluorescently labeling therapeutic mAb candidates during screening and lead selection phases utilizes Zenon™ antibody labeling technology. The Zenon labeling reagent is a fluorophore-, biotin-, or enzyme-labeled Fab fragment directed against the Fc portion of a primary IgG antibody (Figure 2). As such, their binding does not interfere with the complementarity-determining region (CDR) of the antibody, and therefore affinity and specificity are unaffected. Zenon technology can be used to rapidly generate Alexa Fluor, allophycocyanin (APC), or R-phycoerythrin (R-PE) antibody conjugates, which can then serve as flow cytometry detection reagents for target validation across cell line panels or for characterization of transient and stable cell lines. Although the interaction between the Zenon Fab fragment and the primary antibody is noncovalent, the binding is sufficient to allow detection of internalization and trafficking of the conjugates within cells. Figure 3 shows an example of SK-BR-3 breast cancer cells treated for 30 minutes with Herceptin™ (trastuzumab, Roche) labeled using the Zenon Alexa Fluor 594 Human IgG Labeling Reagent. Both cell surface–bound and internalized pools of labeled Herceptin bound to its target HER2/ErbB2 are clearly visualized. Trafficking of the Herceptin/Zenon Alexa Fluor 594 complex to lysosomes is confirmed by colocalization with LysoTracker™ Deep Red Reagent.

The SiteClick™ Antibody Labeling Kits provide a modular, click chemistry–mediated method for enzymatically labeling essentially any antibody on its heavy chain N-linked glycans—far from the CDR—providing excellent reproducibility from labeling to labeling and from antibody to antibody. This site-selective strategy is especially important when labeling monoclonal antibodies that contain lysine residues in or around the CDR, as labeling of these sites with amine-reactive dyes can disrupt antigen binding. While SiteClick Antibody Labeling Kits are currently available for creating your own fluorescent R-PE and Qdot™ conjugates, the SiteClick technology can be applied to your monoclonal antibody using a variety of Molecular Probes dyes through our Custom Services. For more information, please email your inquiry to Thermo Fisher Scientific Custom Services .

Table 1. Molecular Probes™ antibody labeling kits.

Amount Antibody labeling kit * Notes
10–20 µg APEX™ Alexa Fluor™ Antibody Labeling Kits
(also available with Pacific Blue™ and Oregon Green™ 488 dyes)
  • Labeled IgG antibodies ready to use in 2.5 hr (~15 min hands-on time)
  • Solid-phase labeling using a pipette tip preloaded with resin
  • Efficient labeling in serum, ascites fluid, or hybridoma supernatants
1–20 µg Zenon™ Antibody Labeling Kits
  • Labeled IgG antibodies ready to use in 10 min
  • Isotype-specific, noncovalent labeling with dyes, haptens, or enzymes
  • Efficient labeling in serum, ascites fluid, or hybridoma supernatants
20–100 µg Alexa Fluor™ Microscale Protein Labeling Kits
(also available for biotin labeling)
  • Labeled proteins ready to use in 2 hr (~30 min hands-on time)
  • Optimized for proteins between 10 and 150 kDa
  • Stabilizing proteins must be removed from sample before labeling
100 µg Alexa Fluor™ Monoclonal Antibody Labeling Kits
(also available with Pacific Blue™ and Pacific Orange™ dyes)
  • Labeled proteins ready to use in 90 min (~15 min hands-on time)
  • Optimized for small-scale labeling of any protein >40 kDa
  • Stabilizing proteins must be removed from sample before labeling
100–125 µg SiteClick™ Antibody Labeling Kits
(available with R-PE and Qdot™ labels)
  • Labeling occurs over 3 days, with ~3 hr hands-on time
  • Modular, click chemistry–mediated method for enzymatically labeling an antibody on its heavy chain N-linked glycans, far from the antigen-binding site
1 mg Alexa Fluor™ Protein Labeling Kits
(also available with Pacific Blue™, Pacific Orange™, fluorescein, Oregon Green™ 488, and Texas Red™ dyes and the biotin hapten)
  • Labeled proteins ready to use in 2 hr (~30 min hands-on time)
  • Optimized for large-scale labeling of IgG antibodies (~150 kDa)
  • Stabilizing proteins must be removed from sample before labeling
0.5–3.0 mg SAIVI™ Alexa Fluor™ Antibody Labeling Kits
  • Labeled proteins ready to use in 90 min (~10 min hands-on time)
  • Control for optimal degree of labeling for Alexa Fluor near-IR dyes
  • Produces azide-free conjugates for in vivo imaging applications
Figure 2. Humanized or fully human IgG antibodies can be noncovalently coupled with fluorescent dyes using Zenon antibody labeling technology. Unconjugated antibodies are incubated with fluorescently labeled Fab fragments directed against the Fc portion of a human IgG antibody. This labeling reaction leaves the complementarity-determining region (CDR) of the target antibody intact and free from obstruction, while providing a consistent degree of labeling (DOL) of 3 to 5 Fab molecules per primary IgG antibody. Zenon™ antibody labeling technology is also available for mouse and rabbit IgG antibodies.  
Figure 3. Immunodetection using Herceptin labeled with Zenon Alexa Fluor 594 Human IgG Labeling Reagent. Herceptin™ was labeled with Zenon™ Alexa Fluor™ 594 Human IgG Labeling Reagent, producing a DOL of approximately 3 Fab fragments per mAb. SK-BR-3 breast carcinoma cells, which highly express HER2/ ErbB2, were treated with 1 μg/mL Herceptin/Zenon Alexa Fluor 594 complex and 50 nM LysoTracker™ Deep Red Reagent in complete medium for 30 min at 37°C. Live cells were imaged to detect (A) surface-bound as well as internalized Herceptin/Zenon Alexa Fluor 594 complex (pseudocolored green) and (B) Lysotracker Deep Red labeling (pseudocolored red). (C) Within the 30 min incubation period, it is apparent that some of the Herceptin/ Zenon complex has reached lysosomes, shown by colocalization with the LysoTracker Deep Red fluorescence.  

Tools for following antibody internalization and trafficking

As therapeutic mAbs interact with membrane-bound targets at the cell surface, they are often internalized via clathrin-mediated endocytosis and trafficked to lysosomes, where they are catabolized by proteolytic enzymes. This internalization and lysosomal degradation can have a profound influence on the pharmacokinetic properties of therapeutic mAbs, including accelerated clearance from the circulation that could significantly affect the dosing regimen. In other cases, notably for ADCs, lysosomal degradation of the peptide linker between the antibody and the cytotoxic payload is required for efficacy. Thus, there is an emerging appreciation for understanding the disposition of therapeutic mAbs after they interact with their cellular targets.

A powerful method to visualize trafficking of therapeutic mAbs in live cells takes advantage of the pH gradient that exists between the extracellular space and vesicular compartments of the endolysosomal pathway. Upon internalization, mAbs are exposed to an increasingly acidic environment as they are trafficked from early endosomes to late endosomes and eventually to lysosomes (Figure 4). pHrodo™ Green and pHrodo™ Red dyes are minimally fluorescent at neutral and basic pH values; these dyes, however, show increasing fluorescence emission with decreasing pH conditions (Figure 5). Thus, conjugates made using pHrodo dyes can be used to directly determine when mAbs are trafficked to endosomes and lysosomes in live cells using microscopy or flow cytometry [5]. Alternately, pHrodo and Zenon technologies can be combined to produce a reagent that enables rapid labeling of mAbs with a pH-sensitive dye for internalization and trafficking studies. In Figure 6, SK-BR-3 cells were treated for 30 minutes at 37°C with Herceptin labeled using a custom Zenon pHrodo Red Human IgG Labeling Reagent. The internalized Herceptin/Zenon pHrodo complex is readily observed in live cells, and trafficking to lysosomes is confirmed by colocalization with LysoTracker Deep Red Reagent. Note that there is no fluorescence emitted by membrane-bound Herceptin/Zenon complex, consistent with the pH-dependent emission properties of pHrodo dyes and their conjugates. For more information on the Zenon pHrodo Labeling Reagents, please send your inquiry to

An alternate method for discriminating internalized versus cell surface–bound pools of therapeutic mAbs is to utilize the fluorescence-quenching capabilities of rabbit anti–Alexa Fluor 488 dye antibody. When primary antibodies conjugated to Alexa Fluor 488 dye are added to live cells under conditions that promote surface binding and internalization, the cells can then be incubated with saturating concentrations of anti–Alexa Fluor 488 dye antibody. This treatment effectively quenches the fluorescence from the antibody bound to the surface without attenuating the signal from the internalized fluorescent label. Herter and colleagues combined this approach with flow cytometric analysis to demonstrate that different anti-CD20 variants display different internalization dynamics that correlate with drug efficacy [6].




Figure 4. Gradient of acidification in vesicles of the endosome–lysosome pathway. As antibodies are taken up by cells from the extracellular space (pH 7.4), they are sequestered within early endosomes (pH ~6.3), then trafficked to late endosomes (pH ~5.5) and finally to lysosomes (pH ~4.7), where they are degraded. In some cases, antibodies may escape lysosomal degradation by being targeted to recycling endosomes, which direct them back to the cell surface for exocytosis.



Figure 5. pH-dependent fluorescence emission profiles of pHrodo™ Red dextran and pHrodo™ Green dextran conjugates.

Figure 6. Immunodetection using Herceptin labeled with a custom Zenon pHrodo Red Human IgG Labeling Reagent. pHrodo™ Red was conjugated to Fab fragments directed against the Fc portion of a human IgG antibody in order to generate a custom Zenon™ reagent for directly detecting Herceptin™ trafficking to lysosomes. SK-BR-3 cells were incubated with 1 μg/mL Herceptin/Zenon pHrodo Red complex and 50 nM LysoTracker™ Deep Red Reagent in complete medium for 30 min at 37°C. (A) Herceptin/Zenon pHrodo Red complex labeling (pseudocolored green). (B) LysoTracker Deep Red labeling (pseudocolored red). (C) Merged image. Images acquired from live cells clearly demonstrate the localization of internalized Herceptin/Zenon pHrodo Red complex within lysosomes. Because pHrodo Red is essentially nonfluorescent at neutral pH values, the Herceptin/Zenon pHrodo Red complex bound to the cell surface is not detected in this experiment.  

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