A general process for the development of peptide-based immunoassays for monoclonal antibodies

Abstract

PurposeMonoclonal antibodies (mAb) are an important and growing class of cancer therapeutics, but pharmacokinetic analyses have in many cases been constrained by the lack of standard and robust pharmacologic assays. The goal of this project was to develop a general method for the production of immunoassays that can measure the levels of therapeutic monoclonal antibodies in biologic samples at relevant concentrations.MethodsAlemtuzumab and rituximab are monoclonal approved for the treatment of B-cell malignancies and were used as a model system. Phage-displayed peptide libraries were screened for peptide sequences recognized by alemtuzumab (anti-CD52) or rituximab (anti-CD20). Synthetic biotinylated peptides were used in enzyme-linked immunosorbent assays (ELISA). Peptides directly synthesized on polymer resin beads were used in an immunofluorescent-based assay.ResultsPeptide mimetope sequences were recovered for both mAb and confirmed by competitive staining and kinetic measurements. A peptide-based ELISA method was developed for each. The assay for rituximab had a limit of detection of 4 μg/ml, and the assay for alemtuzumab had a limit of detection of 1 μg/ml. Antibody-specific staining of peptide conjugated beads could be seen in a dose-dependent manner.ConclusionPhage-displayed peptide libraries can be a source of highly specific mimetopes for therapeutic mAb. The biotinylated forms of those peptides are compatible with conventional ELISA methods with sensitivities comparable to other assay methods and sufficient for pharmacological studies of those mAb given at high dose. The process outlined here can be applied to any mAb to enable improved pharmacokinetic analysis during the development and clinical use of this class of therapies.Electronic supplementary materialThe online version of this article (doi:10.1007/s00280-009-1240-1) contains supplementary material, which is available to authorized users.

EGFR-binding peptide: a patent evaluation of WO2014002836

“…Structural analysis of selected ligands can provide new information about ligand--target interactions and can be useful in further drug discovery and development. Currently, there are several kinds of phage display library system such as M13, T7, and so on [23][24][25][26]. In this study, the authors employed Ph.D-12 phage display library system, which was proved to be a better method for this screening of EGFR-binding peptides compared with Ph.C7C phage display library system.…”

Individualized Dosing of Therapeutic Monoclonal Antibodies—a Changing Treatment Paradigm?

“…MAb TDM tests that utilize mimetope peptides as capture or detection reagents in ELISA and lateral flow immunoassay (POC device) are also being developed. Peptides specific for rituximab and alemtuzumab were originally described (102,103), and many others have been developed. These peptides, which are referred to as Veritopes, are selected via phage display against mAb targets (novel or biosimilar) or other proteins, such as receptor fusions.…”

Development of a Sensitive Bead-Based Assay for Enhanced Monoclonal Antibody Detection

“…Alemtuzumab and rituximab antigen mimetic peptides sequences were generated by phage-displayed peptide library screening and synthesized on 10μm Tentagel beads as described previously (14). Automated handling and multiplexing capability were enabled on the bead platform by the trapping of nanoparticles within the polystyrene matrix of the bead.…”

“…Beads were then measured on a BD FACSCalibur (Becton, Dickinson and Company, Franklin Lakes, NJ) and the data was analyzed and gated using FlowJo (Treestar, Ashland, OR) software. Limit of detection was performed using the protocol described previously (14). indicating a time dependent non-specific background response in addition to the serum dependent background response changes (Figure 2).…”

“…A determination of the limit of detection was performed on a serum sample with 7 spiked alemtuzumab concentrations 0, 0.1, 0.5, 1, 2, 4, 8ug/ml on 3 different days in quadruplicate and was determined to be 0.5ug/ml, as determined by the lower 95 th percentile above zero. An ELISA based assay limit of detection based on a 6 samples performed over 4 different days was determined to be 1ug/ml (14).…”