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. 2022 Jun 24:2022:3748315.
doi: 10.1155/2022/3748315. eCollection 2022.

Noninvasive Evaluation of EGFR Expression of Digestive Tumors Using 99mTc-MAG3-Cet-F(ab')2-Based SPECT/CT Imaging

Dai Shi #  1   2   3   4 , Yiqiu Zhang #  1   2   3   4 , Zhan Xu  1   2   3   4 , Zhan Si  1   2   3   4 , Yuan Cheng  1   2   3   4 , Dengfeng Cheng  1   2   3   4 , Guobing Liu  1   2   3   4
Affiliations

Noninvasive Evaluation of EGFR Expression of Digestive Tumors Using 99mTc-MAG3-Cet-F(ab')2-Based SPECT/CT Imaging

Dai Shi et al. Mol Imaging. .

Abstract

Purpose: This study is aimed at investigating the feasibility of cetuximab (Cet) F(ab')2 fragment- (Cet-F(ab')2-) based single photon emission tomography/computed tomography (SPECT/CT) for assessing the epidermal growth factor receptor (EGFR) expression in digestive tumor mouse models.

Methods: Cet-F(ab')2 was synthesized using immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) protease and purified with protein A beads. The product and its in vitro stability in normal saline and 1% bovine serum albumin were analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The EGFR expression in the human colon tumor cell line HT29 and the human stomach tumor cell line MGC803 were verified using western blotting and immunocytochemistry. Cet-F(ab')2 was conjugated with 5(6)-carboxytetramethylrhodamine succinimidyl ester to demonstrate its binding ability to the MGC803 and HT29 cells. Cet-F(ab')2 was conjugated with NHS-MAG3 for 99mTc radiolabeling. The best imaging time was determined using a biodistribution assay at 1, 4, 16, and 24 h after injection of the 99mTc-MAG3-Cet-F(ab')2 tracer. Furthermore, 99mTc-MAG3-Cet-F(ab')2 SPECT/CT was performed on MGC803 and HT29 tumor-bearing nude mice.

Results: HT29 cells had low EGFR expression while MGC803 cell exhibited the high EGFR expression. Cet-F(ab')2 and intact cetuximab showed similar high binding ability to MGC803 cells but not to HT29 cells. Cet-F(ab')2 and 99mTc-MAG3-Cet-F(ab')2 showed excellent in vitro stability. The biodistribution assay showed that the target to nontarget ratio was the highest at 16 h (17.29 ± 5.72, n = 4) after tracer injection. The 99mTc-MAG3-Cet-F(ab')2-based SPECT/CT imaging revealed rapid and sustained tracer uptake in MGC803 tumors rather than in HT29 tumors with high image contrast, which was consistent with the results in vitro.

Conclusion: SPECT/CT imaging using 99mTc-MAG3-Cet-F(ab')2 enables the evaluation of the EGFR expression in murine EGFR-positive tumors, indicating the potential utility for noninvasive evaluation of the EGFR expression in tumors.

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Conflict of interest statement

The authors declare that there is no conflict of interest regarding this article.

Figures

Figure 1
Figure 1
Schematic diagram illustrating the synthesis of 99mTc-MAG3-Cet-F(ab′)2. Please note that this figure only illustrates the synthesis of the radiolabel, and not that only one MAG3 molecule attaches to one cetuximab moiety.
Figure 2
Figure 2
Evaluation of the EGFR expression in MGC803 and HT29 tumor cells. Western blots (a) and immunofluorescence of tumor cell (b) showed a higher EGFR expression level in MGC803 cells compared to HT29 cells.
Figure 3
Figure 3
Verification of Cet-F(ab′)2 on its molecular weight, binding affinity to EGFR positive cells, and in vitro stability. (a) SDS-PAGE showing that the molecular weight of Cet-F(ab′)2 and cetuximab is 100 kDa and 150 kDa, respectively. (b) Immunocytochemistry showing high binding ability of Cet-F(ab′)2 and cetuximab to MGC803 cells. (c) SDS-PAGE illustrating the excellent stability of Cet-F(ab′)2 after incubation in PBS and 1% BSA over 24 h.
Figure 4
Figure 4
The HPLC results showing ultraviolet profiles of MAG3-Cet, MAG3-Cet-F(ab′)2 and MAG3-Cet-Fc. (a) The HPLC result of MAG3-Cet. (b) The HPLC result of MAG3-Cet-F(ab′)2 and MAG3-Cet-Fc after digesting MAG3-Cet with IdeS protease. (c) The HPLC result of the mixture from (b) but after reaction with protein A beads. The residual MAG3-Cet and most of the MAG3-Cet-Fc were removed by protein A beads. (d) The HPLC result of the mixture of (a) and (c).
Figure 5
Figure 5
Characterization of 99mTc-MAG3-Cet-F(ab′)2 in vitro. (a) The radio-HPLC result of 99mTc-MAG3-Cet-F(ab′)2. (b) Stability assay of 99mTc-MAG3-Cet-F(ab′)2 in 1% BSA and NS. (c) Competition binding assay between 99mTc-MAG3-Cet-F(ab′)2 and unlabeled cetuximab to MGC803 cells (d, e). Representative saturation binding curve of 99mTc-MAG3-Cet-F(ab′)2 binding to MGC803 cells (d) and HT29 cells (e), indicating its higher affinity to MGC803 cells (Bmax = 5.68 × 10−19 mol ligands/cell, Kd = 0.6147 nM), compared to HT29 cells (Bmax = 1.66 × 10−19 mol ligands/cell, Kd = 1.008 nM).
Figure 6
Figure 6
In vitro biodistribution and SPECT/CT imaging. (a) Biodistribution assay demonstrating that 99mTc-MAG3-Cet-F(ab′)2 uptake in MGC803 tumors peaked at 16 h after injection. (b) 99mTc-MAG3-Cet-F(ab′)2 SPECT/CT imaging showing significant different radionuclide uptake between an MGC803 tumor and a HT29 tumor (arrow). (c) Immunofluorescence showing the higher EGFR expression in MGC803 tumor slices compared with HT29 tumor slices. (d) Relationship between SPECT/CT image and immunofluorescence.
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