High-Throughput Generation of Bispecific Binding Proteins by Sortase A-Mediated Coupling for Direct Functional Screening in Cell Culture.

Fabio Andres,Martin Schwill,Ykelien L Boersma,Andreas Plückthun

doi:10.1158/1535-7163.mct-19-0633

Fabio Andres, Martin Schwill + Show 2 more

Open Access

https://doi.org/10.1158/1535-7163.mct-19-0633

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Abstract

High-throughput construction of multivalent binders and subsequent screening for biological activity represent a fundamental challenge: A linear increase of monovalent components translates to the square of possible bivalent combinations. Even high-efficiency cloning and expression methods become limiting when thousands of bispecific binders need to be screened for activity. In this study, we present an in vitro method for the efficient production of flexibly linked bispecific binding agents from individually expressed and purified monovalent binders. We established a sortase A-mediated coupling reaction to generate bispecific designed ankyrin repeat proteins (DARPins), with an optimized reaction maximizing the bivalent coupling product with low levels of monovalent side-products. These one-pot reaction mixtures could be used directly, without further purification, in cell-based assays. We generated a matrix of 441 different bispecific DARPins against the extracellular domains of the cancer-associated receptors EGFR, ErbB2, ErbB3, ErbB4, EpCAM, and c-MET and screened on two different ErbB2-positive cancer cells lines for growth-inhibitory effects. We identified not only known but also novel biologically active biparatopic DARPins. Furthermore, we found that the cancer cell lines respond in a highly reproducible and defined manner to the treatment with the 441 different bivalent binding agents. The generated response profiles can thus be used for functional characterization of cell lines because they are strongly related to the cell line-specific surface receptor landscape. Thus, our method not only represents a robust tool for screening and lead identification of bispecific binding agents, but additionally offers an orthogonal approach for the functional characterization of cancer cell lines.

Highlights

Multivalent, multidomain, and multispecific binding proteins are commonly found in nature [1], where they regulate the spatial and temporal proximity of thousands of protein–protein interactions [2]
Multivalent binding proteins, such as adaptor or scaffold proteins, enable specific signaling events between their target proteins while preventing others. This specificity is encoded in their primary sequence [3], and overexpression of particular multivalent adaptor proteins is known to induce oncogenic transformation [4]. Extracellular growth factors such as hepatocyte growth factor (HGF; ref. 5) or FGF [6] are prominent proto-oncogenes, which stimulate the activation of receptor tyrosine kinases (RTK) through intermolecular bridging and stabilization of the RTK–dimer interactions, which are found frequently upregulated in various types of cancers and are associated with the development of cancer drug resistance [6, 7]
We directly screened for the biological effect of the bispecific constructs on HER2þ cancer cell lines in high-throughput cell viability assays, and we identified a set of novel bispecific designed ankyrin repeat proteins (DARPins) molecules that can either block cancer cell proliferation or, significantly stimulate it

Summary

Introduction

Multivalent, multidomain, and multispecific binding proteins are commonly found in nature [1], where they regulate the spatial and temporal proximity of thousands of protein–protein interactions [2]. Thereby, they control various signaling cascades by binding to their targets. Multivalent binding proteins, such as adaptor or scaffold proteins, enable specific signaling events between their target proteins while preventing others This specificity is encoded in their primary sequence [3], and overexpression of particular multivalent adaptor proteins is known to induce oncogenic transformation [4]. Extracellular growth factors such as hepatocyte growth factor (HGF; ref. 5) or FGF [6] are prominent proto-oncogenes, which stimulate the activation of receptor tyrosine kinases (RTK) through intermolecular bridging and stabilization of the RTK–dimer interactions, which are found frequently upregulated in various types of cancers and are associated with the development of cancer drug resistance [6, 7].

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