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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.

ABSTRACT: 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.

SUBMITTER: Schaller TH

PROVIDER: S-EPMC7325320 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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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.

Schaller Teilo H TH Foster Matthew W MW Thompson J Will JW Spasojevic Ivan I Normantaite Deimante D Moseley M Arthur MA Sanchez-Perez Luis L Sampson John H JH

Journal of proteome research 20190719 8

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 EGFRvII ...[more]

PMID: 31267741

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Project description:Purpose: Conventional therapy for malignant glioma fails to specifically target tumor cells. In contrast, substantial evidence indicates that if appropriately redirected, T cells can precisely eradicate tumors. Here we report the rational development of a fully human bispecific antibody (hEGFRvIII-CD3 bi-scFv) that redirects human T cells to lyse malignant glioma expressing a tumor-specific mutation of the EGFR (EGFRvIII).Experimental Design: We generated a panel of bispecific single-chain variable fragments and optimized design through successive rounds of screening and refinement. We tested the ability of our lead construct to redirect naïve T cells and induce target cell-specific lysis. To test for efficacy, we evaluated tumor growth and survival in xenogeneic and syngeneic models of glioma. Tumor penetrance following intravenous drug administration was assessed in highly invasive, orthotopic glioma models.Results: A highly expressed bispecific antibody with specificity to CD3 and EGFRvIII was generated (hEGFRvIII-CD3 bi-scFv). Antibody-induced T-cell activation, secretion of proinflammatory cytokines, and proliferation was robust and occurred exclusively in the presence of target antigen. hEGFRvIII-CD3 bi-scFv was potent and target-specific, mediating significant lysis of multiple malignant glioma cell lines and patient-derived malignant glioma samples that heterogeneously express EGFRvIII. In both subcutaneous and orthotopic models, well-engrafted, patient-derived malignant glioma was effectively treated despite heterogeneity of EGFRvIII expression; intravenous hEGFRvIII-CD3 bi-scFv administration caused significant regression of tumor burden (P < 0.0001) and significantly extended survival (P < 0.0001). Similar efficacy was obtained in highly infiltrative, syngeneic glioma models, and intravenously administered hEGFRvIII-CD3 bi-scFv localized to these orthotopic tumors.Conclusions: We have developed a clinically translatable bispecific antibody that redirects human T cells to safely and effectively treat malignant glioma. On the basis of these results, we have developed a clinical study of hEGFRvIII-CD3 bi-scFv for patients with EGFRvIII-positive malignant glioma. Clin Cancer Res; 24(15); 3611-31. ©2018 AACR.

Project description:We recently reported the development of a fully-human, CD3-binding bispecific antibody for immunotherapy of malignant glioma. To translate this therapeutic (hEGFRvIII-CD3- bi-scFv) to clinical trials and to help further the translation of other similar CD3-binding therapeutics, some of which are associated with neurologic toxicities, we performed a good laboratory practice (GLP) toxicity study to assess for potential behavioral, chemical, hematologic, and pathologic toxicities including evaluation for experimental autoimmune encephalomyelitis (EAE). To perform this study, male and female C57/BL6 mice heterozygous for the human CD3 transgene (20/sex) were allocated to one of four designated groups. All animals were administered one dose level of hEGFRvIII-CD3 bi-scFv or vehicle control. Test groups were monitored for feed consumption, changes in body weight, and behavioral disturbances including signs of EAE. Urinalysis, hematologic, and clinical chemistry analysis were also performed. Vehicle and test chemical-treated groups were humanely euthanized 48 hours or 14 days following dose administration. Complete gross necropsy of all tissues was performed, and selected tissues plus all observed gross lesions were collected and evaluated for microscopic changes. This included hematoxylin-eosin histopathological evaluation and Fe-ECR staining for myelin sheath enumeration. There were no abnormal clinical observations or signs of EAE noted during the study. There were no statistical changes in food consumption, body weight gain, or final body weight among groups exposed to hEGFRvIII-CD3 bi-scFv compared to the control groups for the 2- and 14-day timepoints. There were statistical differences in some clinical chemistry, hematologic and urinalysis endpoints, primarily in the females at the 14-day timepoint (hematocrit, calcium, phosphorous, and total protein). No pathological findings related to hEGFRvIII-CD3 bi-scFv administration were observed. A number of gross and microscopic observations were noted but all were considered to be incidental background findings. The results of this study allow for further translation of this and other important CD3 modulating bispecific antibodies.

Dataset Information

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

Publications

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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