Glycoprotein Receptor CEACAM5-Targeted Intraoperative Molecular Imaging Tracer in Non-Small Cell Lung Cancer

Abstract

Background: Intraoperative molecular imaging has emerged as a potential tool in addressing challenges faced during lung cancer surgery by localizing small lesions, ensuring negative margins, and identifying synchronous cancers. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) glycoprotein has emerged as a potential target in fluorescent labeling of non-small cell lung cancer given the high antigen density in tumor cells and absence of expression in normal parenchyma. The goal of our study was to determine whether anti-CEACAM5 targeted near-infrared fluorochrome could be a suitable target in non-small cell lung cancer.

Methods: The CEACAM5 expression was evaluated in AB-12 (known negative control), HT29 (known positive control), and H460 (non-small cell lung cancer) cell lines by polymerase chain reaction. SGM-101, a CEACAM5 antibody, coupled with a BM-104 near-infrared fluorescent tracer was evaluated with dose escalation, in vitro cellular localization, and immunofluorescence microscopy. Subsequently, in vivo validation was performed in 52 athymic nude xenografts.

Results: Polymerase chain reaction analysis demonstrated 3000x relative expression of CEACAM5 in HT-29 cells compared with AB-12. The H460 cells showed 1000x relative expression compared with AB12 (P < .05). Both HT29 and H460 cells showed tracer internalization with signal to background ratio of 4.5 (SD 0.34) whereas there was minimal uptake by AB12 cells with signal to background ratio 1.1 (SD 0.1; P < .05). There was linear fluorescence increase with increasing tracer dosing in receptor expressing cell lines. In preclinical models, HT-29 and H460 cells lines produced near-infrared fluorescence with average tumor to background ratio of 3.89 (SD 0.25) irrespective of tumor size compared with no fluorescence by AB12 tumors (P < .05). The CEACAM5 expressing tumors had excellent dye uptake compared with AB12 tumors.

Conclusions: CEACAM5 serves as a possible receptor for targeted intraoperative molecular imaging resections in lung cancer. This study sets a path for evaluation of CEACAM5 targets in future clinical trials.

Figure 1:

TCGA analysis of cancer type and CEACAM5 mRNA expression with associated mutation frequency for each cancer type.

Figure 2:

(A) qPCR analysis of CEACAM5 relative mRNA expression in HT29, H460, A549, and AB12 cell lines. (B) RT-PCR analysis of AB12, HT29, H460, and A549 cell lines showing detection of CEACAM5 in HT29 and H460 cells with relative chemiluminescence levels displayed below. Analyses were performed in triplicates.

Figure 3:

Flow cytometry analysis for primary CEACAM5 antibody binding to AB12, H460, and HT29 cells with no binding observed in AB12 cells. Anti-CEACAM5 to irrelevant Ab GMean ratios were 3.6, 134.5 and 109.6 for AB12, HT29 and H460 cells, respectively.

Figure 4:

Immunofluorescence microscopy of SGM-101 in HT29, AB12, H460 cells showing dye uptake and internalization in CEACAM5+ cells.

Figure 5:

(A): Immunofluorescence NIR microscopy shows concentration dependent increase in cellular uptake and fluorescence in HT29 and H460 cell lines with absence of fluorescence detected in AB12 cell lines at 24 hours. (B): Concentration kinetics quantifying observed fluorescence intensity in the cell lines with Kd of 12 pM demonstrating high specificity of the tracer. (C): Near Infrared emission comparison of SGM-101 to ICG at concentrations of 1 uM to 100nM.

Figure 6:

Time course and site-specific fluorescence of SGM-101 in HT29 positive control cells.

Figure 7:

(A): Mouse xenografts of HT29, H460, and AB12 cell lines demonstrates SGM-101 localization to CEACAM5 expressing cells which is concordant with in-vitro findings. (B) SGM-101 fluorescence emission in positive and negative controls as well as organ biodistribution in both the control groups demonstrating expected uptake of the tracer in the GI tract and CEACAM5+ xenograft while no uptake or fluorescence was detected in the lung parenchyma of both controls. (C): Histopathologic assessment of CEACAM5 targeted SGM-101 demonstrating tumor specific localization.

Figure 8:

Representative mice images of H460 tumor resection with positive and negative margin detection.