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J Proteome Res. 2019 Jul 19. doi: 10.1021/acs.jproteome.9b00145. [Epub ahead of print]

Pharmacokinetic Analysis of a Novel Human EGFRvIII:CD3 Bispecific Antibody in Plasma and Whole Blood Using a High-Resolution Targeted Mass Spectrometry Approach.

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Preston Robert Tisch Brain Tumor Center , Duke University Medical Center , Durham , North Carolina , United States.
Department of Neurosurgery , Duke University Medical Center , Durham , North Carolina , United States.
Department of Pathology , Duke University Medical Center , Durham , North Carolina , United States.
Duke Proteomics and Metabolomics Shared Resource, Duke Center for Genomic and Computational Biology , Duke University , Durham , North Carolina , United States.
Duke Cancer Institute PK/PD Core Laboratory , Durham , North Carolina , United States.
Department of Medicine , Duke University School of Medicine , Durham , North Carolina , United States.


Bispecific single chain antibody fragments (bi-scFv) represent an emerging class of biotherapeutics. We recently developed a fully human bi-scFv (EGFRvIII:CD3 bi-scFv) with the goal of redirecting CD3-expressing T cells to recognize and destroy malignant, EGFRvIII-expressing glioma. In mice, we showed that EGFRvIII:CD3 bi-scFv effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma. Here, we developed a targeted assay for pharmacokinetic (PK) analysis of EGFRvIII:CD3 bi-scFv, a necessary step in the drug development process. Using microflow liquid chromatography coupled to a high resolution parallel reaction monitoring mass spectrometry, and data analysis in Skyline, we developed a bottom-up proteomic assay for quantification of EGFRvIII:CD3 bi-scFv in both plasma and whole blood. Importantly, a protein calibrator, along with stable isotope-labeled EGFRvIII:CD3 bi-scFv protein, were used for absolute quantification. A PK analysis in a CD3 humanized mouse revealed that EGFRvIII:CD3 bi-scFv in plasma and whole blood has an initial half-life of ∼8 min and a terminal half-life of ∼2.5 h. Our results establish a sensitive, high-throughput assay for direct quantification of EGFRvIII:CD3 bi-scFv without the need for immunoaffinity enrichment. Moreover, these pharmacokinetic parameters will guide drug optimization and dosing regimens in future IND-enabling and phase I studies of EGFRvIII:CD3 bi-scFv.


absolute quantification; biotherapeutic; bispecific antibody; glioma; mass spectrometry; parallel reaction monitoring; pharmacokinetic analysis; stable isotope labeled protein; targeted proteomics

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