# A Method for High‐Throughput Screening of Monoclonal Antibody Internalization Using a DNA/Protein Molecular Staple

## TL;DR

The paper introduces a new method to efficiently measure how monoclonal antibodies bind to and are taken up by cells, without needing chemical modifications.

## Contribution

A one-pot, chemical-free method using a DNA/protein staple to screen monoclonal antibody internalization into cells is developed.

## Key findings

- High antibody binding does not always result in high internalization into cells.
- Internalization efficiency depends on both the antibody and receptor characteristics.
- The method allows rapid screening of multiple lymphocyte receptors for binding and internalization.

## Abstract

The use of monoclonal antibodies (mAbs) as therapeutics or for targeted delivery to specific cells is reliant on understanding mAb‐receptor interactions. Of particular importance is the relationship between mAb binding and internalization into cells. Internalization can be determined using molecular sensors; however, the challenges associated with functionalizing and purifying mAbs with these sensors often mean internalization is not assessed. To address this, we have developed a simple, one‐pot approach that requires no chemical modifications of the mAbs that efficiently quantifies mAb binding and internalization into cells. This is achieved by developing a protein staple that binds a DNA based internalization sensor to mAbs. This sensor is used to screen mAb binding and internalization into T cell and B cell receptors present on human peripheral blood mononuclear cells. The differences between cell binding and receptor internalization can be rapidly assessed for an array of lymphocyte receptors. These results demonstrate that high binding does not necessarily correlate to high uptake into the cell or high internalization efficiency of a receptor. Receptor internalization efficiency is a product of intrinsic characteristics of both the receptor and mAb, highlighting the need to screen for these functions to improve mAb therapeutics.

Internalization of antibodies plays a critical role in targeted drug delivery and the activity of antibody therapeutics. A simple, one‐pot, chemical modification free approach is developed to quantify antibody internalization into cells.