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

Sanchez AB, Nguyen T, Dema-Ala R, Kummel AC, Kipps TJ, Messmer BT - Cancer Chemother. Pharmacol. (2010)

Bottom Line: Peptide mimetope sequences were recovered for both mAb and confirmed by competitive staining and kinetic measurements.Phage-displayed peptide libraries can be a source of highly specific mimetopes for therapeutic mAb.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.

View Article: PubMed Central - PubMed

Affiliation: Moores Cancer Center, University of California San Diego, La Jolla, CA, 92093-0815, USA.

ABSTRACT

Purpose: Monoclonal 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.

Methods: Alemtuzumab 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.

Results: Peptide 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 microg/ml, and the assay for alemtuzumab had a limit of detection of 1 microg/ml. Antibody-specific staining of peptide conjugated beads could be seen in a dose-dependent manner.

Conclusion: Phage-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.

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Alemtuzumab bead-based detection. Different concentrations of labeled alemtuzumab were analyzed by flow cytometry using pC1-1T conjugated beads
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Fig4: Alemtuzumab bead-based detection. Different concentrations of labeled alemtuzumab were analyzed by flow cytometry using pC1-1T conjugated beads

Mentions: An advantage to using peptides as the primary capture agent in solid phase immunoassays is that they can be directly synthesized on a variety of substrates. To demonstrate this, the rituximab and alemtuzumab peptides were synthesized on 10-μm diameter TentaGel beads, hereafter known as pCp-1T and pRTX-10T. TentaGel, a PEG polystyrene co-polymer, is a common solid phase synthesis support material, and commercially prepared peptides are typically cleaved from the surface of such beads. Specific cognate antibody binding was confirmed by flow cytometry and fluorescent microscopy (Fig. 3 in “Supplementary material”). Alemtuzumab was titrated into normal human serum and incubated with the pCp-1T beads. After washing and addition of a fluorescent secondary anti-human IgG antibody, the fluorescence on the beads was quantitated by flow cytometry. The fluorescent signals correlated with the alemtuzumab concentration, with very little background from serum alone (Fig. 4).Fig. 4


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

Sanchez AB, Nguyen T, Dema-Ala R, Kummel AC, Kipps TJ, Messmer BT - Cancer Chemother. Pharmacol. (2010)

Alemtuzumab bead-based detection. Different concentrations of labeled alemtuzumab were analyzed by flow cytometry using pC1-1T conjugated beads
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2921063&req=5

Fig4: Alemtuzumab bead-based detection. Different concentrations of labeled alemtuzumab were analyzed by flow cytometry using pC1-1T conjugated beads
Mentions: An advantage to using peptides as the primary capture agent in solid phase immunoassays is that they can be directly synthesized on a variety of substrates. To demonstrate this, the rituximab and alemtuzumab peptides were synthesized on 10-μm diameter TentaGel beads, hereafter known as pCp-1T and pRTX-10T. TentaGel, a PEG polystyrene co-polymer, is a common solid phase synthesis support material, and commercially prepared peptides are typically cleaved from the surface of such beads. Specific cognate antibody binding was confirmed by flow cytometry and fluorescent microscopy (Fig. 3 in “Supplementary material”). Alemtuzumab was titrated into normal human serum and incubated with the pCp-1T beads. After washing and addition of a fluorescent secondary anti-human IgG antibody, the fluorescence on the beads was quantitated by flow cytometry. The fluorescent signals correlated with the alemtuzumab concentration, with very little background from serum alone (Fig. 4).Fig. 4

Bottom Line: Peptide mimetope sequences were recovered for both mAb and confirmed by competitive staining and kinetic measurements.Phage-displayed peptide libraries can be a source of highly specific mimetopes for therapeutic mAb.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.

View Article: PubMed Central - PubMed

Affiliation: Moores Cancer Center, University of California San Diego, La Jolla, CA, 92093-0815, USA.

ABSTRACT

Purpose: Monoclonal 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.

Methods: Alemtuzumab 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.

Results: Peptide 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 microg/ml, and the assay for alemtuzumab had a limit of detection of 1 microg/ml. Antibody-specific staining of peptide conjugated beads could be seen in a dose-dependent manner.

Conclusion: Phage-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.

Show MeSH
Related in: MedlinePlus