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

J Proteome Res. 2019 Aug 2;18(8):3032-3041. doi: 10.1021/acs.jproteome.9b00145. Epub 2019 Jul 19.


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.

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

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antibodies, Bispecific / blood*
  • Antibodies, Bispecific / pharmacokinetics
  • Antibodies, Bispecific / therapeutic use
  • CD3 Complex / blood*
  • CD3 Complex / pharmacokinetics
  • CD3 Complex / therapeutic use
  • Cell Line, Tumor
  • Chromatography, Liquid
  • ErbB Receptors / blood*
  • ErbB Receptors / pharmacokinetics
  • ErbB Receptors / therapeutic use
  • Glioblastoma / blood*
  • Glioblastoma / immunology
  • Glioblastoma / therapy
  • Humans
  • Mass Spectrometry
  • Mice
  • Proteomics / methods
  • T-Lymphocytes / immunology
  • Xenograft Model Antitumor Assays


  • Antibodies, Bispecific
  • CD3 Complex
  • epidermal growth factor receptor VIII
  • ErbB Receptors