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

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Pharmacokinetic Analysis of a Novel Human EGFRvIII:CD3 Bispecific Antibody in Plasma and Whole Blood Using a High-Resolution Targeted Mass Spectrometry Approach.
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2019-07-19 , DOI: 10.1021/acs.jproteome.9b00145
Teilo H Schaller _{1,

2,

3} , Matthew W Foster ₄ , J Will Thompson ₄ , Ivan Spasojevic _{5,

6} , Deimante Normantaite ₁ , M Arthur Moseley ₄ , Luis Sanchez-Perez _{1,

2} , John H Sampson _{1,

2,

3}

Affiliation

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.

中文翻译：

使用高分辨率靶向质谱法在血浆和全血中检测新型人EGFRvIII：CD3双特异性抗体的药代动力学。

双特异性单链抗体片段（bi-scFv）代表了一类新兴的生物治疗药物。我们最近开发了一种完全人类的bi-scFv（EGFRvIII：CD3 bi-scFv），其目标是重定向表达CD3的T细胞以识别和破坏表达EGFRvIII的恶性神经胶质瘤。在小鼠中，我们表明EGFRvIII：CD3 bi-scFv有效治疗原位患者源性恶性神经胶质瘤和同基因胶质母细胞瘤。在这里，我们开发了针对EGFRvIII：CD3 bi-scFv的药代动力学（PK）分析的靶向分析，这是药物开发过程中的必要步骤。使用微流液相色谱与高分辨率并行反应监测质谱联用，并在Skyline中进行数据分析，我们开发了一种自下而上的蛋白质组学测定方法，用于定量测定血浆和全血中的EGFRvIII：CD3 bi-scFv。重要的是，蛋白质校正剂连同稳定的同位素标记的EGFRvIII：CD3 bi-scFv蛋白一起用于绝对定量。CD3人源化小鼠的PK分析显示，血浆和全血中的EGFRvIII：CD3 bi-scFv的初始半衰期约为8分钟，终末半衰期约为2.5 h。我们的结果建立了灵敏的高通量检测方法，可直接定量检测EGFRvIII：CD3 bi-scFv，而无需进行免疫亲和富集。此外，这些药代动力学参数将指导EGFRvIII：CD3 bi-scFv的未来IND启用和I期研究中的药物优化和给药方案。5小时我们的结果建立了灵敏的高通量检测方法，可直接定量检测EGFRvIII：CD3 bi-scFv，而无需进行免疫亲和富集。此外，这些药代动力学参数将指导EGFRvIII：CD3 bi-scFv的未来IND启用和I期研究中的药物优化和给药方案。5小时我们的结果建立了灵敏的高通量检测方法，可直接定量检测EGFRvIII：CD3 bi-scFv，而无需进行免疫亲和富集。此外，这些药代动力学参数将指导EGFRvIII：CD3 bi-scFv的未来IND启用和I期研究中的药物优化和给药方案。

更新日期：2019-07-20

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