An EGFR targeted PET imaging probe for the detection of colonic adenocarcinomas in the setting of colitis

Abstract

Colorectal cancer is a serious complication associated with inflammatory bowel disease, often indistinguishable by screening with conventional FDG PET probes. We have developed an alternative EGFR-targeted PET imaging probe that may be used to overcome this difficulty, and successfully assessed its utility for neoplastic lesion detection in preclinical models. Cetuximab F(ab')2 fragments were enzymatically generated, purified, and DOTA-conjugated. Radiolabeling was performed with (67)Ga for cell based studies and (64)Cu for in vivo imaging. Competitive binding studies were performed on CT26 cells to assess affinity (KD) and receptors per cell (Bmax). In vivo imaging using the EGFR targeted PET probe and (18)F FDG was performed on CT26 tumor bearing mice in both control and dextran sodium sulfate (DSS) induced colitis settings. Spontaneous adenomas in genetically engineered mouse (GEM) models of colon cancer were additionally imaged. The EGFR imaging agent was generated with high purity (> 98%), with a labeling efficiency of 60 ± 5% and ≥99% radiochemical purity. The KD was 6.6 ± 0.7 nM and the Bmax for CT26 cells was 3.3 ± 0.1 × 10(6) receptors/cell. Target to background ratios (TBR) for CT26 tumors compared to colonic uptake demonstrated high values for both (18)F-FDG (3.95 ± 0.13) and the developed (64)Cu-DOTA-cetuximab-F(ab')2 probe (4.42 ± 0.11) in control mice. The TBR for the EGFR targeted probe remained high (3.78 ± 0.06) in the setting of colitis, while for (18)F FDG, this was markedly reduced (1.54 ± 0.08). Assessment of the EGFR targeted probe in the GEM models demonstrated a correlation between radiotracer uptake in spontaneous colonic lesions and the EGFR staining level ex vivo. A clinically translatable PET imaging probe was successfully developed to assess EGFR. The imaging agent can detect colonic tumors with a high TBR for detection of in situ lesions in the setting of colitis, and opens the possibility for a new approach for screening high-risk patients.

Keywords: EGFR; Positron emission tomography (PET) imaging; colorectal cancer; molecular imaging; mouse models.; ulcerative colitis.

Figure 1

A schematic overview of the EGFR targeted PET probe synthesis and characterization. (A) Enzymatic fragmentation of whole antibody and conjugation of F(ab')2 fragment with bifunctional chelator. (B) FPLC chromatograms of whole IgG before digestion, after digestion, and after protein A purification. (C) SDS-PAGE analysis of enzymatic digestion of cetuximab IgG. (i) Whole antibody before digestion; (ii) F(ab')₂ and Fc bands after digestion of whole antibody; (iii) F(ab')₂ band after protein A purification. (D) Radiolabeling and purification of F(ab')2-DOTA conjugate with ⁶⁷Ga. (E) Direct (saturation) radioligand binding to CT26 murine colorectal cancer cells of ⁶⁴Cu-DOTA-cetuximab-F(ab')2, in the absence (total binding; TB) or presence (non-specific binding; NSB) of excess (x 20 times) unlabeled cetuximab IgG. Specific binding (SB) was calculated by subtraction of NSB from TB. Curves were fit to a 1-site receptor-binding model. ◆= TB; ■= NSB; ▴= SB. (F) Western blot of EGFR expression in CT26 and HCT-116 colon cancer cell lines with beta-actin as control, demonstrate EGFR overexpression.

Figure 2

PET imaging of EGFR expression in BALB/c mice at 24 hr post injection of ⁶⁴Cu-DOTA-cetuximab-F(ab′)2 or ⁶⁴Cu-DOTA-cetuximab-F(ab')2 after treatment with a 1.5 mg blocking dose of cetuximab. A) Example images without and with blocking. Red circles highlight site of implanted CT26 tumor allograft B) Comparison of MeanSUV values of ⁶⁴Cu-DOTA-cetuximab-F(ab′)2 without and with blocking. Each bar represents SUVmean ± SEM; n=4 for each group, and the symbol * denotes P<0.05. C) Comparison of tumor to muscle ratios of ⁶⁴Cu-DOTA-cetuximab-F(ab′)2 without and with blocking. Each bar represents SUVmean ± SEM; n=4 for each group, and the symbol * denotes P<0.05.

Figure 3

Assessment of symptoms and histological findings of DSS-treated and control mice. BABL/c mice were subjected to 3% DSS for seven days and followed with normal drinking water for three days. (A) Body weight changes following DSS induction of colitis, reported as mean ± SEM (n=4 mice/group) and the symbol * denotes P<0.05. (B) microPET-CT scans were obtained before and after DSS treatment. Before DSS treatment, PET showed only low level of physiologic ¹⁸F-FDG uptake in the colon of healthy controls. In contrast, on day nine, clearly elevated ¹⁸F-FDG colonic uptake is noted (red arrow). (C) Histological appearance of the normal colonic mucosa of healthy controls with intact crypts and architecture (control) and DSS-induced colitis showing inflammatory infiltration (arrow head), mucin loss (arrow) and crypt architectural disarray (dashed arrow). Magnification x20 and scale bar represents 100 μm.

Figure 4

Comparison of PET imaging with ¹⁸F-FDG and ⁶⁴Cu-DOTA-cetuximab-F(ab′)2 in CT26 tumor-bearing mice with and without DSS-induced colitis. (A) PET-CT images were performed 1 hr after ¹⁸F-FDG and 24 hr after ⁶⁴Cu-DOTA-cetuximab-F(ab')2 administration. Representative images from a mouse are shown, with n=4 mice imaged in each group. Red squares indicate the location of tumors and blue squares indicate location of DSS-induced colitis. (B) Comparison of tumor to colon ratios at 24 hr post-injection. Each bar represents SUVmean ± SEM; n=4 for each group. The symbol * denote P<0.05 and the difference is not significant (P=0.22) for ⁶⁴Cu-DOTA-cetuximab-F(ab′)2. The dash line verifies TBR=1 (no contrast).

Figure 5

Correlation between EGFR targeted PET imaging and EGFR immunohistochemistry. (A1) PET imaging of Apc^CKOP53^flox/flox mouse with ⁶⁴Cu-DOTA-cetuximab-F(ab′)2 at 24 hr. (A2) The digital photograph of colon and the colon fused with the ex-vivo PET image. (A3) Corresponding EGFR expression patterns of different colon sections numbered in A2 are confirmed with immunohistochemistry (magnification x20). (B1) Representative images of decay corrected PET image of Apc^{LoxP/ LoxP}Msh2^null/LoxP mutant mice after the injection of ⁶⁴Cu-DOTA-cetuximab-F(ab′)2 at 24 hr. (B2) The digital photograph of colon and the colon fused with the ex-vivo PET image. (B3) EGFR expression patterns of different colon sections numbered in B2 are confirmed with immunohistochemistry (magnification x20). Scale bar represents 50 μm.

Figure 6

H&E and IHC analysis were examined to compare the histological changes in colons from healthy, DSS-treated, GEM models and allograft (CT26 tumor). IHC results demonstrate that there is no significant EGFR expression in healthy and DSS-treated colons, while there is strong staining in allograft and GEM model colonic lesions. (A) Representative H&E tissue sections from (a1) healthy colon, (a2) DSS-treated colon, (a3) CT26 allograft, (a4) Apc^CKOP53^flox/flox mutant mouse colon, (a5) Apc^LoxP/LoxPMsh2^null/LoxPmutant mouse colon. (B) IHC analysis of EGFR from tissues of (b1) healthy colon, (b2) DSS-treated colon, (b3) CT26 allograft, (b4) Apc^CKOP53^flox/flox mutant mouse colon, (b5) Apc^{LoxP/ LoxP}Msh2^null/LoxPmutant mouse colon. Scale bar represents 50 μm.