Affimers as Anti-Idiotypic Affinity Reagents for Pharmacokinetic Analysis of Biotherapeutics

Abstract

Therapeutic antibodies are the fastest growing class of drugs in the treatment of cancer, and autoimmune and inflammatory diseases that require the concomitant development of assays to monitor therapeutic antibody levels. Here, we demonstrate that the use of Affimer nonantibody binding proteins provides an advantage over current antibody-based detection systems. For four therapeutic antibodies, we used phage display to isolate highly specific anti-idiotypic Affimer reagents, which selectively bind to the therapeutic antibody idiotype. For each antibody target the calibration curves met US Food and Drug Administration criteria and the dynamic range compared favorably with commercially available reagents. Affimer proteins therefore represent promising anti-idiotypic reagents that are simple to select and manufacture, and that offer the sensitivity, specificity and consistency required for pharmacokinetic assays.

Enrichment and Liquid Chromatography–Mass Spectrometry Analysis of Trastuzumab and Pertuzumab Using Affimer Reagents

“… 23 Affimer reagents have been used in different applications such as molecule detection, biosensing, and cell imaging with promising results. 20 , 24 − 29 An interesting study involving Affimer reagents targeted against four monoclonal antibodies in a sandwich ELISA produced results that met Food and Drug Administration (FDA) requirements for accuracy and precision when compared to commercial, antibody-based assay kits. 24 …”

“… 20 , 24 − 29 An interesting study involving Affimer reagents targeted against four monoclonal antibodies in a sandwich ELISA produced results that met Food and Drug Administration (FDA) requirements for accuracy and precision when compared to commercial, antibody-based assay kits. 24 …”

“…20,24−29 An interesting study involving Affimer reagents targeted against four monoclonal antibodies in a sandwich ELISA produced results that met Food and Drug Administration (FDA) requirements for accuracy and precision when compared to commercial, antibody-based assay kits. 24 Here, we describe an approach to enrich trastuzumab and pertuzumab from human plasma in combination with mass spectrometry analysis. Affimer reagents were initially selected from a library of about 10 10 clones based on their capacity to specifically bind to trastuzumab or pertuzumab without crossreactivity to other human immunoglobulin G (IgGs).…”

“…Affimer reagents possess similar affinity and specificity as antibody-based binders but are smaller, produced more easily in bacterial expression systems, stable at high temperatures, and over a wide pH range . Affimer reagents have been used in different applications such as molecule detection, biosensing, and cell imaging with promising results. ,− An interesting study involving Affimer reagents targeted against four monoclonal antibodies in a sandwich ELISA produced results that met Food and Drug Administration (FDA) requirements for accuracy and precision when compared to commercial, antibody-based assay kits …”

Engineering Protein Switches for Rapid Diagnostic Tests

“…Nature does provide alternative binding proteins to antibodies, such as periplasmic binding proteins, but they are limited to known examples and have only been used in protein switches targeting certain small molecules. ,,,, The scope can be broadened using antibody mimetic nonimmunoglobulin binding proteins that consist of a scaffold and variable peptide regions. , They are selected with high affinity and specificity for new analytes by display methods, so development is quick and straightforward. , Counter-selection against competing molecules and affinity maturation can be used to further increase specificity and affinity. , Such proteins are also small, nonglycosylated, and cysteine-free, , so within protein switches they maintain the geometric benefits of antibody fragments while being easier to recombinantly manufacture. This makes them particularly attractive recognition elements and many, including Affimers, DARPINS, and monobodies, have been successfully utilized in protein switch sensors for varied analytes. ,,, …”