A new EGFR and c-Met bispecific NIR-II fluorescent probe for visualising colorectal cancer and metastatic lymph nodes

Background: The aim of the study was to increase the specificity and targeting of tumour imaging, targeting molecules that enable the simultaneous recognition and binding of multiple tumour-associated receptors. We constructed a NIR-II fluorescence probe based on a bispecific antibody to epidermal growth factor receptor (EGFR) and cellular mesenchymal–epithelial transition factor (c-Met) for visualising colorectal cancers (CRCs) and metastatic lymph nodes. Methods: The expression of EGFR and c-Met in tumour and metastatic lymph node specimens from patients with CRC was examined using immunohistochemistry. The EGFR and c-Met bispecific antibody (Rybrevant) was labelled, and its cell-specific binding ability was assessed using laser confocal microscopy. Subcutaneous CRC and orthotopic tumour models were constructed to evaluate the fluorescence imaging of the probe in vivo. To assess the performance of Rybrevant-IRDye800CW in the differential diagnosis of metastatic lymph nodes, a CRC lymph node metastasis model was constructed using human CRC cells implanted in mouse claw pads. Finally, surgically resected CRC tumours and lymph node specimens were incubated with Rybrevant-IRDye800CW for fluorescence NIR-II imaging to evaluate the efficacy of Rybrevant-IRDye800CW for preclinical visualisation. Findings: The combined expression rate of EGFR and c-Met in CRC and metastatic lymph nodes was significantly higher than the single-target expression rate. The bispecific probe Rybrevant-IRDye800CW was successfully synthesised, and its fluorescence signal could be extended up to 1600 nm using NIR-II imaging. Cell incubation experiments showed that the fluorescence intensity of Rybrevant-IRDye800CW was strongly correlated with EGFR and c-Met overexpression of the cells. NIR-II in vivo fluorescence imaging showed that double-positively expressing subcutaneous tumours significantly uptook Rybrevant-IRDye800CW after tail vein injection of the probe, which rapidly accumulated within the tumours in about 6 h. In EGFR and or c-Met blockade assays, subcutaneous tumours showed weaker uptake of Rybrevant-IRDye800CW. Similarly, Rybrevant-IRDye800CW was specifically identified in orthotopic CRC and lymph node metastasis models, with all orthotopic tumours showing high tumour-to-background ratios in NIR-II imaging. In a NIR-II preclinical study, Rybrevant-IRDye800CW could specifically identify fresh human CRC and its metastatic lymph node tissue. Interpretation: This study confirmed the complementary EGFR and c-Met expression in CRC and its metastatic lymph nodes. Compared to single-target probes, EGFR and c-Met dual-specific fluorescent probes identified CRC and its metastatic lymph nodes using NIR-II imaging. Thus, NIR-II-guided R0 surgery was performed to resect the CRC and metastatic lymph nodes. Fundings: This study was supported by the Beijing Natural Science Foundation (Grant numbers: L222054, 7244517, 4232058, L248026, L232020), National Natural Science Foundation of China (NSFC) ( 92059207, 92359301, 92259303, 62027901, 81930053, 81227901, U21A20386), CAS Youth Interdisciplinary Team (JCTD-2021-08), and the Fundamental Research Funds for the Central Universities (Grant no. JK2024-2-35-02).