Construction and preclinical immnoPET imaging of a gallium-68 radiolabeled specific nanobody for targeting CD147 detection in pan-cancer

Introduction: The extracellular matrix metalloproteinase inducer CD147, which is significantly over-expressed in various solid tumors but scarcely found in normal tissues, offers an optimal target for molecular imaging and targeted therapy. Nanobodies are the smallest fragments derived from antibodies and are easily modified for conjugation with functional reagents. They possess ideal characteristics suitable for PET imaging applications, such as a rapid clearance rate and excellent distribution curves, allowing for same-day imaging with sufficient contrast. In this study, we developed a novel CD147-targeted nanobody radiotracer, ⁶⁸Ga-NOTA-Mal-NB147, guiding the noninvasive detection of CD147 in pan-cancer.

Methods: In this study, we utilized the dual-function chelator NOTA-Mal for the specific site-specific labeling of the CD147-targeted nanobody NB147 with nuclide ⁶⁸Ga. Then the probe was purified by size exclusion chromatography column (PD-10), and its quality control and in vitro stability were determined by a radioactive thin layer chromatography scanner (radio-TLC). The in vitro stability of the ⁶⁸Ga-NOTA-Mal-NB147 in normal saline and 5% human serum albumin (5% HSA) was analyzed. The molecular weights of CD147 nanobody (NB147) were determined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Flow cytometry (FCM), Western blot (WB), Immunohistochemistry (IHC), and immunofluorescence (IF) were used to verify the expression of CD147 on the surface of human colon cancer cells LS174T, human pancreatic cancer cells BXPC3, human liver cancer cells SMMC-7721 and human gastric cancer cells NCI-N87, and screen high and low expression models. The affinity of ⁶⁸Ga-NOTA-Mal-NB147 to CD147 protein was determined by radio-ELISA experiment, and ⁶⁸Ga-NOTA-Mal-NB147 was used for cell uptake experiments in vitro. In vivo, biodistribution studies of KM mice and tumor model mice were performed by tail vein injection. The above cell lines and balb/c nude mice were used to establish subcutaneous colon cancer, pancreatic cancer, liver cancer and gastric cancer models respectively, and immunoPET imaging was performed at 0.5 h, 1 h, 2 h, and 4 h.

Results: The labeled ⁶⁸Ga-NOTA-Mal-NB147 was purified by PD-10. The results showed that the radiochemical purity was over 98% and maintained over 95% in both 0.01 moL/L PBS and 10 % HSA for more than 4 h. The molecular weight of NB147 was 17.1 kDa as measured by MALDI-TOF-MS. FCM, WB, IHC and IF showed that the cell lines LS174T, BXPC3 and SMMC-7721 expressed CD147 highly, while NCI-N87 showed low expression of CD147. The radio-ELISA indicated that ⁶⁸Ga-NOTA-Mal-NB147 had high binding affinity to CD147. The cell uptake test showed that there was a significant difference in ⁶⁸Ga-NOTA-Mal-NB147 uptake between CD147 high-expression cells and CD147 low-expression cells (P<0.01). The results of the biodistribution experiment show that the probe has a higher uptake in the kidneys and is primarily metabolized and excreted from the body through urine. In contrast, the uptake levels in normal liver, lung, muscle, stomach, and intestines are all at relatively low levels and gradually decrease with increasing injection time. The imaging results show that in CD147-positive tumor models, the probe accumulates at the tumor sites of LS174T, BXPC3, and SMMC-7721. However, in CD147-negative tumor models, no significant uptake was observed at the tumor site of NCI-N87. Simultaneously, the co-injection of an excess amount of cold antibody significantly reduces the uptake of the probe in LS174T tumors, indicating the specific targeting binding of ⁶⁸Ga-NOTA-Mal-NB147 to CD147 high-expression tumor models.

Conclusions: We successfully validated one specific CD147 nanobody, NB147, and the derived immunoPET probe ⁶⁸Ga-NOTA-Mal-NB147 showed precise visualization for accurate diagnosis of CD147-expressing lesions in pan-cancer.