Abstract 2443: MR imaging in human glioma tumor xenograft models using enzyme-conjugated anti-EGFRvIII-specific antibody fragments

Abstract

Abstract A truncated and constitutively active form of the EGF receptor variant III (EGFRvIII) is a major determinant of tumor growth and poor prognosis in glioblastoma multiforme [1]. To test a system for targeted imaging of EGFRvIII we investigated the retention of peroxidase-generated products of a paramagnetic substrate diTyr-DTPAGd in U87αEGFR orthotopic human glioma xenografts. F(ab’)2 fragments of EMD72000 mAb were conjugated to deglycosylated horseradish peroxidase (HRP) and glucose oxidase (GOX) and used as a self-complementing enzymatic signal amplification system [2] for EGFRvIII targeted imaging. We anticipated that receptor expression sites will exhibit a prolonged MR signal enhancement due to the formation of polymerized products of diTyr-DTPAGd oxidation by mAb conjugates [2]. F(ab’)2 fragments of mAb were linked to enzymes using bisaromatic hydrazone bonds and purified by HPLC. The conjugates were characterized in U87αEGFR cell culture and optimal ratios of HRP and GOX conjugates were determined to provide the maximum signal with low cytotoxicity. F(ab’)2-GOX and F(ab’)2-HRP were also modified with NHS-MAG3 for radiolabeling. U87αEGFR cells were stereotaxically implanted in the brains of RNU rats. Ten days after tumor implantation, each animal was imaged using T1-weighted spin-echo MRI (TR/TE=700ms/8.2ms) at 3T on two occasions: Day 1 - images were acquired after IV injection of 0.1 mmol/kg diTyr-DTPAGd over a 1.5h period; Day 2 - anti-EGFRvIII conjugates (100 µg mAb/animal) were injected IV, and then 4h later 0.1 mmol/kg diTyr-DTPAGd was injected IV followed by image acquisition for 2h. Both 99mTc-labeled conjugates showed specific binding to U87αEGFR cells in vitro and in vivo as demonstrated using SPECT/CT. Cell-binding and internalization studies showed that 80% conjugates were internalized at 37°C. MR T1-weighted images showed significantly higher enhancement and longer retention of the MR signal on Day 2 in rats pre-injected with conjugates compared to Day 1 over the same time period due to the conjugate co-localization at the EGFRvIII target sites. The washout of the contrast agent was best modeled using a biexponential decay as compared to Gli36αEGFR-bearing rats [4]. U87αEGFR tumors showed biexponential MR signal decay on both days. Administration of EGFRvIII-targeted mAb conjugates resulted in specific binding to U87αEGFR cells of which at least ∼20% remained on the surface enabling the reaction with the contrast agent. The biexponential decay constants on Day 2 for both Gli36αEGFR and U87αEGFR animals were not significantly different indicating a similarity of conjugate accumulation in both tumor models which resulted in a similar bimodal washout of the contrast agent.