In this Solution, NextGenRnD reports a novel platform technology aimed at the identification of antibodies (Abs) that upon interaction with an antigen, present on mammalian cell surface, trigger an internalization event. These antibodies will be referred to as internalizing (i.e., iAb). The antigen present on the surface of a mammalian cell can be a receptor, a protein, or any other molecule that can be recognized and bound by antibody. Likewise, Fab, scFv, and any other form derived from Ab structure or peptide can be used instead of Ab.
Library-based phage display technology had been used to successfully identify internalizing antibodies and their fragments (Fab, scFv, etc.) for nearly two decades now, whereas approaches based on other technologies were not as successful. Abs, Fabs, and scFvs not displayed on phage cannot be screened in the context of complex mixed populations due to high copy number requirement by downstream analytic methods. In particular, each antibody should be present in large quantities (1012–1015 molecules) within a library to enable its subsequent identification by mass spectrometry, whereas a single infectious phage particle is sufficient for displayed antibody identification. Thus, phage display-based technology should be considered as the most advanced and the most sensitive technology for the identification of internalizing antibodies.
Commercially available internalization assays or kits for phage display libraries are not available. However, several companies provide custom discovery service to identify internalizing antibodies. Available data (such as phage display library characteristics, bacterial hosts, type of phages used, etc.) strongly suggests that the experimental approach used by these custom discovery service providers is essentially identical to the one used in academia.
The NEXTGENRND® Solution No. 1 consists of two parts. In the first part, the critical analysis of current experimental approach (termed Prototype Protocol), used for the identification of internalizing antibodies and based on phage display, will be presented. In the second part, the performed analysis will be used to modify the current library-based internalizing antibody screening approach. The resulting Optimal Protocol is faster, more efficient, and more versatile.