Matching the decay half-life with the biological half-life: ImmunoPET imaging with (44)Sc-labeled cetuximab Fab fragment

Abstract

Scandium-44 (t1/2 = 3.9 h) is a relatively new radioisotope of potential interest for use in clinical positron emission tomography (PET). Herein, we report, for the first time, the room-temperature radiolabeling of proteins with (44)Sc for in vivo PET imaging. For this purpose, the Fab fragment of Cetuximab, a monoclonal antibody that binds with high affinity to epidermal growth factor receptor (EGFR), was generated and conjugated with N-[(R)-2-amino-3-(para-isothiocyanato-phenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N',N″,N″-pentaacetic acid (CHX-A″-DTPA). The high purity of Cetuximab-Fab was confirmed by SDS-PAGE and mass spectrometry. The potential of the bioconjugate for PET imaging of EGFR expression in human glioblastoma (U87MG) tumor-bearing mice was investigated after (44)Sc labeling. PET imaging revealed rapid tumor uptake (maximum uptake of ∼12% ID/g at 4 h postinjection) of (44)Sc-CHX-A″-DTPA-Cetuximab-Fab with excellent tumor-to-background ratio, which might allow for same day PET imaging in future clinical studies. Immunofluorescence staining was conducted to correlate tracer uptake in the tumor and normal tissues with EGFR expression. This successful strategy for immunoPET imaging of EGFR expression using (44)Sc-CHX-A″-DTPA-Cetuximab-Fab can make clinically translatable advances to select the right population of patients for EGFR-targeted therapy and also to monitor the therapeutic efficacy of anti-EGFR treatments.

Figure 1

Generation of Cetuximab-Fab and its characterization. (A) Schematic diagram for Cetuximab-Fab generation from intact antibody, conjugation, and radiolabeling. The figures are not drawn to scale. (B) SDS-PAGE to confirm the purity of Cetuximab-Fab (lane 1, ladder; lane 2, intact Cetuximab; lane 3, unpurified Cetuximab-Fab after papain digestion; and lane 4, purified Cetuximab-Fab after passing through Protein A column). (C) Mass spectrometry of Cetuximab-Fab (∼49.9 kDa).

Figure 2

Flow cytometry in U87MG (high EGFR expression) and Caco-2 (low EGFR expression) cells confirms the EGFR specificity and affinity of Cetuximab-Fab.

Figure 3

Serial PET imaging of EGFR expression. (A) Serial coronal PET images at 0.5, 2, 4, and 6 h p.i. of (I) ⁴⁴Sc–CHX-A″-DTPA–Cetuximab-Fab in U87MG tumor-bearing mice (targeted), (II) ⁴⁴Sc–CHX-A″-DTPA-denatured-Cetuximab-Fab in U87MG tumor-bearing mice (non-targeted), (III)⁴⁴Sc–CHX-A″-DTPA–Cetuximab-Fab after treatment with a 2 mg blocking dose of Cetuximab before injection in U87MG tumor-bearing mice, and (IV) ⁴⁴Sc–CHX-A″-DTPA–Cetuximab-Fab in Caco-2 tumor-bearing mice (negative control). (B) Representative PET/CT images of U87MG tumor-bearing mouse at 4 h p.i. of ⁴⁴Sc–CHX-A″-DTPA–Cetuximab-Fab. Arrowheads indicate tumors.

Figure 4

Quantitative region-of-interest (ROI) analysis of the PET data. Time–activity curves of the liver, tumor, blood, kidney, and muscle following intravenous injection of (A) ⁴⁴Sc–CHX-A″-DTPA–Cetuximab-Fab in U87MG tumor-bearing mice (targeted), (B) ⁴⁴Sc–CHX-A″-DTPA-denatured-Cetuximab-Fab in U87MG tumor-bearing mice (non-targeted), (C) ⁴⁴Sc–CHX-A″-DTPA–Cetuximab-Fab in U87MG tumor-bearing mice after treatment with a 2 mg blocking dose of Cetuximab (blocking), (D) ⁴⁴Sc–CHX-A″-DTPA–Cetuximab-Fab in Caco-2 tumor-bearing mice (negative control). (E) Comparison of tracer uptake in the tumors among all four groups.

Figure 5

Biodistribution of ⁴⁴Sc–CHX-A″-DTPA–Cetuximab-Fab in U87MG tumor-bearing mice (targeted), ⁴⁴Sc–CHX-A″-DTPA–Cetuximab-Fab in U87MG tumor-bearing mice after treatment with a blocking dose of Cetuximab (blocking), and ⁴⁴Sc–CHX-A″-DTPA–Cetuximab-Fab in Caco-2 tumor-bearing mice (negative control) at 6 h p.i. (n = 3).