Internalizing Antibody Screening

An antibody or peptide that binds a receptor on the mammalian cell surface can be drawn into the cell through receptor-mediated endocytosis. Once inside, it becomes a vehicle: the same binder that recognizes the target receptor can carry a drug, toxin, enzyme, or nucleic acid to the intracellular space. Antibodies selected for this behavior — internalizing antibodies — are therefore useful wherever the payload must reach the inside of the cell rather than act only at the surface.

We screen libraries for clones that not only bind the receptor but are taken up by the cell. The selection separates true internalizers from surface-only binders by distinguishing phage that have entered the cell from phage that merely remains attached outside it.

Figure 1. Schematic illustration of the concept of the antibody internalization and degradation assay. (Source: Li Y, et al. 2015)

How Internalization Is Selected

• Bind at the surface – incubate the library with receptor-bearing cells so binders attach to the receptor.
• Allow uptake – resuspend the cells in medium and incubate at a suitable temperature so receptor-bound phage antibodies are internalized.
• Remove the external signal – wash the cells with buffer to strip off phage that are still bound at the surface but never entered.
• Recover the internalized fraction – collect the phage that crossed the membrane and amplify it for the next round.
• Identify the clones – sequence the enriched internalizers and express the candidates for confirmation.

Display Systems Used

Receptors Covered

Internalizing-antibody screening can be run against a range of cell-surface receptors. Commonly requested targets include EGFR, CTLA4, HER2, CD20, CD3, CD59, and CD33. Where a receptor is not on this list, the same workflow is applied to the receptor of interest provided it is expressed on a suitable cell line.

What You Receive

• Internalizing clones – binders confirmed to enter the cell, not only to attach at the surface.
• Sequence data – the variable regions of the selected antibodies or fragments.
• Expressed candidates – reformatted molecules available for uptake and functional confirmation.
• Documentation – selection rounds and the evidence distinguishing internalized from surface-bound phage.

Applications

• Targeted payload delivery – conjugating a drug, toxin, enzyme, or nucleic acid to an internalizing binder.
• Therapeutic lead discovery – internalizing antibodies for intracellular targets or cargo delivery.
• Cell-entry research – tools to study receptor trafficking and uptake.
• Diagnostic and imaging – binders that report on receptor expression and behavior.