Targeted signal-amplifying enzymes enhance MRI of EGFR expression in an orthotopic model of human glioma

Abstract

Epidermal growth factor receptor (EGFR) imaging in brain tumors is essential to visualize overexpression of EGFRvIII variants as a signature of highly aggressive gliomas and to identify patients that would benefit from anti-EGFR therapy. Seeking imaging improvements, we tested a novel pretargeting approach that relies on initial administration of enzyme-linked anti-EGFR monoclonal antibodies (mAb; EMD72000) followed by administration of a low-molecular-weight paramagnetic molecule (diTyr-GdDTPA) retained at the site of EGFR mAb accumulation. We hypothesized that diTyr-GdDTPA would become enzyme activated and retained on cells due to binding to tissue proteins. In support of this hypothesis, mAb-enzyme conjugates reacted with both membrane-isolated wild-type (wt) EGFR and EGFRvIII, but they bound primarily to EGFRvIII-expressing cells and not to EGFRwt-expressing cells. In vivo analysis of magnetic resonance (MR) tumor signal revealed differences in MR signal decay following diTyr-GdDTPA substrate administration. These differences were significant in that they suggested differences in substrate elimination from the tissue which relied on the specificity of the initial mAb binding: a biexponential signal decay was observed in tumors only upon preinjection with EGFR-targeted conjugates. Endpoint MRI in this setting revealed detailed images of tumors which correlated with immunohistochemical detection of EGFR expression. Together, our findings suggest an improved method to identify EGFRvIII-expressing gliomas in vivo that are best suited for treatment with therapeutic EGFR antibodies.

Figure 1

A– Synthesis of peroxidase-reducing paramagnetic substrate di(tyramido)-DTPA(Gd); B– Reaction of diTyr-GdDTPA with the peroxidase/glucose oxidase enzyme pair conjugated to anti-EGFR mAb.

Figure 2

A– SDS-PAGE (4–15% gradient) of anti-EGFR mAb (EMD72000) conjugation products or with deglycosylated enzymes: HRP (37 kD, lanes 1 and 2) and with GOX (69 kD subunit, lanes 3, 4) Lanes 1 and 3- before and 2,4- after the purification of conjugates; B– immunoblotting of membrane proteins isolated from Gli36ΔEGFR (Δ) and Gli36wt (WT) cells using mouse monoclonal anti-EGFR antibody C225 or by using HRP-EMD72000 conjugate. EGFR variants are identified on the right; C – titration of the mixture of anti-EGFR mAb-HRP and mAb-GOX on Gli36ΔEGFR cells at the optimized complementing ratio (1:2, w/w); D – binding and internalization of conjugate mixture at the optimized ratio (1:2, w/w) in Gli36ΔEGFR (Δ) and Gli36wt (WT) cells 1,3, 5,7 – cell-surface bound fraction of conjugates; 2,4,6,8 – internalized fraction of conjugates.

Figure 3

A – 3T MR imaging of Gli36ΔEGFR human glioma xenografts without and with pre-injection of EGFR-targeted conjugates. T₁-WT sequential rat brain images depicting enhancement as a function of time post injection of diTyr-GdDTPA; Top row -temporal washout of diTyr-GdDTPA with no conjugate pre-injection (Day 1); Bottom row - washout of diTyr-GdDTPA following pre-treatment with anti-EGFR conjugates (Day 2) in the same slice for the same animal. Time intervals (in minutes) after the injection of diTyr-GdDTPA are shown below; B – Gli36ΔEGFR xenografts without and with pre-injection of EpCAM-targeted conjugates. The images correspond to the same pattern as shown in Panel A; C – MRI and comparative histology. The images were obtained pre-, immediately post- and 1 h post- diTyr-GdDTPA administration; D – Left: Detection of EGFR overexpression using anti-EGFR antibody-digoxigenin/anti-digoxigenin-AP system in the tumor shown in Fig. 3C; Right: Detection of HRP activity in the same tumor on the parallel section using diaminobenzidine staining. Arrowheads point to tumor location; arrows show presence of tumor expansion as microdeposits in normal brain tissue stained for EGFR expression. Bars in B, C = 1 mm.

Figure 4

Immunofluorescent detection of EGF receptor, endothelial cells and mAb conjugate delivery in Gli36ΔEGFR tumors. A – Detection of EGFR expression (green) and blood vessels (anti-CD31, red). The inset shows area of vascularized tumor/brain interface at higher magnification; B – Binding of anti-EGFR-HRP conjugate to cells in the tumor interface after injection. Binding of digoxigenin-labeled anti-HRP antibody (detected by using anti-digoxigenin F(ab′)₂-Cy5.5 conjugate) is shown in red. Nuclei are stained with DAPI. Bar 50 μm. t- tumor, nb- normal brain.

Figure 5

Normalized T₁-WT signal intensities measured in the interface or core regions of the representative Gli36ΔEGFR tumors prior to injection of conjugates (A), or after the pre-injection of either specific anti-EGFR (“EGFR”) or non-specific EpCAM (“EpCAM”) conjugates (B) as a function of time post-diTyr-GdDTPA injection. The signal intensities are normalized as percent change relative to the pre-contrast image. MR signal showed biexponential decay in tumors of animals pre-injected with anti-EGFR conjugates (B, curves 1,2) whereas the MR signal decay curves with no conjugate pre-injection or after the pre-injection of non-specific conjugate (A, curves 1–4, B curves 3,4) showed a single (monoexponential) decay.