Construction of a 89Zr-Labeled Specific Antibody Fragment for the Noninvasive Detection of Mesothelin-Overexpressing Tumors.

Numerous clinical trials on mesothelin (MSLN)-targeted antibodies have provided preliminary evidence supporting their clinical translation, highlighting the importance of patient screening. Although anti-MSLN chimeric antigen receptor T (CAR-T) cell therapy has not been approved by the Food and Drug Administration (FDA) for solid tumor treatment yet, its high expression across various malignancies has made it a major research focus. Prescreening patients suitable for anti-MSLN CAR-T cell therapy may boost both treatment efficacy and safety. The ScFv component of anti-MSLN CAR-T cells retains MSLN targeting capability and serves as a suitable molecular precursor for probe development. In this study, the positron-emitting nuclide 89Zr was utilized to label the antibody fragment MA4 (comprising the ScFv segment of MSLN fused with the human Fc segment), while a mouse monoclonal antibody (Sino Biological, 13128-MM08) served as a control, enabling the development of two immunoPET probes for MSLN imaging. Following purification, 89Zr-MA4 exhibited high radiochemical purity (>99%) and molar activity (24.2-31.9 GBq/μmol) and maintained >95% radiochemical purity over 6 days in both PBS and 5% human serum albumin (HSA). In the MSLN strongly positive LS174T tumor model, the 89Zr-MA4 probe demonstrated expedited tissue penetration and tumor accumulation. The immunohistochemical analysis revealed that LS174T tumors exhibited strong positivity for MSLN, whereas the U87MG group demonstrated only mild expression. Dosage estimation studies have determined that the effective dose of 89Zr-MA4 is 0.142 mSv/MBq, which complies with established safety standards. Overall, both probes exhibit robust in vitro stability and demonstrate feasibility for MSLN-targeted imaging in vivo.