Creative Biolabs currently offers this unique opportunity for our worldwide clients to obtain high-quality service of detecting endocytosis and internalization potencies for lead antibody candidates.
One important mechanism of action of therapeutic antibodies is to induce internalization and turnover of cell surface receptors, resulting in receptor down-modulation as well as antibody endocytosis. This receptor-mediated internalization exhibits great potential for various therapeutic or diagnostic applications. Particularly, due to their abilities of specific binding and intracellular transfer, internalizing antibodies make an excellent vehicle for targeted delivery of drugs, toxins, enzymes or DNA into the cytosol of indicated cells. Therefore, one of the prerequisites for antibodies designed for antibody drug conjugate (ADC) is to induce rapid receptor internalization, so as to ensure cell-specific drug delivery. After internalization, antibody-receptor complexes can either recycle back to the plasma membrane or be degraded by late endosomes and lysosomes. Of note, when at low doses, receptor-mediated internalization activities usually outcompete Fc-mediated recycling, which is based on Fc-FcRn binding and can prevent antibodies from degradation. This can probably explain why internalizing antibodies commonly display shorter half-lives in contrast to antibodies against soluble antigens.
Fig 1. Mechanisms of drug delivery mediated by ADCs. (Senter PD, Sievers EL, 2012)
Given all these appealing features, Creative Biolabs has introduced multiple state-of-the-art techniques to in-depth study the receptor/ antibody internalizing mechanism during the early stage. We offer two assay formats depending on different research objectives.
Phage Display Library Internalization Screening
Creative Biolabs has vast experience in using whole-cell based phage display screening to isolate internalizing antibodies specific for various cell surface receptors, such as EGFR, NTRK2, CTLA-4, HER2, CD20, CD3, CD59, transferrin receptor, and CD33. Methodologies are developed to reduce the number of internalizing phage particles that do not target receptor-specific to exclude phage particles which are merely bound to the cell surface and to selectively identify phage particles that are capable of crosslinking receptors rather than merely binding.
This innovative technology employs a novel reporter tag called fluorogen activated proteins (FAP) to genetically label the indicated membrane receptor, which can exhibit fluorescence after incorporation with particular soluble fluorogens. We successively apply two kinds of fluorogens with the different binding ability and cell permeability. In this way, intracellular and extracellular tags can reveal different colors. Our simple, robust FAP-tag makes a powerful approach for dynamic studies of antibody/receptor internalization and trafficking.
There exists a clear difference in pH when antibodies are transferred from body fluid (neutral) into cellular endosomes and lysosomal vesicles (acidic) upon internalization. Hence, a pH reactive dye agent, which is extremely sensitive to pH changes, can be very helpful to detect internalization activities. We conjugate such pH sensor dyes to antibody candidates (purified or unpurified). After incubation with target cells, internalization activities can then be translated into pH changes-linked fluorescent signals, which can be monitored and analyzed using cell imaging or flow cytometry. This specific approach guarantees minimal background noise because dye agents are cell-impermeable when unconjugated.
Enzymatic Activity
As an alternative, we can label the targeted receptor with a small enzyme fragment, and endosome surface with the complementing enzyme fragment, termed enzyme acceptor (EA). Together, these two fragments can form an active enzyme when the surface receptor is translocated into cytosol via internalization. Therefore, with the proper substrate, the enzymatic activity can act as an indirect indicator of antibody-receptor internalization, offering another rapid, simple, and sensitive strategy.
Creative Biolabs currently possesses a series of GPCR stable cell lines for high-throughput screening of internalizing antibodies. By stable transfection of certain GPCR fused with tGPF, antibody-induced internalization can be easily monitored via FACS. We also have many membrane receptors stable cell lines (e.g. GPCR, ion channel) in stock that are well compatible for various functional studies.
Live Cell Imaging-Based Internalization Assay
In addition, Creative Biolabs has developed a live cell imaging-based internalization assay characterized by high-throughput and high efficiency to screen internalized antibodies. This assay is able to monitor the live cells in a real-time and offers more detailed insights to support global customers' projects.
Our scientists are pleased to share our professional knowledge with customers for assisting their cutting-edge projects. For more detailed information, please feel free to contact us or directly sent us an inquiry.
Reference
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