Development of a Novel Molecular Probe for Visualizing Mesothelin in Tumors

Development of a Molecular Probe for Tumor-Associated Protein Mesothelin Based on PET Imaging Technology

Research Background

In recent years, mesothelin (MSLN) has become a key focus in cancer research and diagnostics. Mesothelin is a membrane-bound glycoprotein highly expressed on the surface of various malignancies, including ovarian, pancreatic, and gastrointestinal cancers, while it is limited to certain serosal tissues in normal adult tissues. Studies have shown that MSLN participates in cell adhesion by binding to the tumor-associated antigen CA125 (cancer antigen 125), a process associated with peritoneal metastasis of ovarian cancer. This molecule has also been linked to chemotherapy resistance and poor prognostic outcomes in cancer patients.

Although serum MSLN can be detected via enzyme-linked immunosorbent assay (ELISA), there is currently no direct, non-invasive method to locate and visualize MSLN expression in tumor tissues. Existing antibody-based imaging studies face various limitations including insufficient specificity and poor tumor tissue penetration. Additionally, therapies such as antibody-drug conjugates or chimeric antigen receptor (CAR) T-cell treatments are often impacted by the release of soluble MSLN from the tumor microenvironment, which reduces therapeutic efficacy. Therefore, developing molecular probes with high specificity and sensitivity to detect MSLN expression in tumors is of great importance.

Article Source

This study, conducted by Yingfang He and colleagues, was published in the European Journal of Nuclear Medicine and Molecular Imaging. It was collaboratively carried out by Fudan University, Guizhou Medical University, and Zentera Therapeutics, and formally published in 2025. The study aims to develop a novel molecular probe based on positron emission tomography (PET) for efficient in vivo visualization of MSLN expression in tumor tissues.

Research Workflow

To develop a molecular probe with high specificity and efficacy for targeting MSLN, the research followed these major steps:

1. Screening and Preparation of VHH Single-Domain Antibodies

The researchers immunized llamas using the 360–597 amino acid fragment of MSLN as the antigen and screened for a low molecular weight single-domain antibody (VHH) named 269-H4. Surface plasmon resonance (SPR) was used to measure its binding kinetics with MSLN, yielding a dissociation constant (Kd) of 0.3 nM, indicating high affinity.

2. Preparation of the Probe Precursor NOTA-269-H4

To enable radiolabeling, the chelator p-SCN-Bn-NOTA was chemically conjugated to 269-H4, resulting in the precursor molecule NOTA-269-H4. Mass spectrometry analysis showed that approximately 27% of the VHH molecules were labeled with a single NOTA chelator. SPR analysis showed that this conjugation did not significantly impact the affinity of the VHH, with the Kd value of NOTA-269-H4 measured at 1.1 nM.

3. Radiolabeling of [68Ga]Ga-NOTA-269-H4

Using a 68Ge/68Ga generator, the researchers manufactured the radioactive isotope 68Ga and successfully synthesized the radiolabeled tracer [68Ga]Ga-NOTA-269-H4 by incubating it at room temperature with NOTA-269-H4. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analyses confirmed that the radiochemical purity (RCP) was greater than 99%, and the product remained stable after storing at room temperature for 90 minutes.

4. Assessment of In Vitro and In Vivo Performance

The tracer [68Ga]Ga-NOTA-269-H4 exhibited high specificity for binding MSLN-positive cells (OVCAR-8 ovarian cancer cell line) as demonstrated by flow cytometry and antibody blocking experiments. In contrast, it showed no significant binding to cells with low or negligible MSLN expression (e.g., SKOV-3).

In vivo, microPET/CT imaging in mice revealed that [68Ga]Ga-NOTA-269-H4 efficiently accumulated in OVCAR-8 tumor-bearing mice, with tumors being clearly visualized. Conversely, minimal radiotracer uptake was observed in SKOV-3 tumor models. In patient-derived xenograft (PDX) models, the tracer further demonstrated high sensitivity and specificity in imaging, while its radioactivity excretion was mainly through the kidneys and bladder.

Study Findings

Through a series of experiments, the researchers obtained the following key results: - The VHH single-domain antibody 269-H4 exhibited high affinity, making it an ideal component for diagnostic molecular probes. - The radiolabeled tracer [68Ga]Ga-NOTA-269-H4 displayed high purity, stability, and effective in vivo performance. - In microPET/CT imaging, the tracer was able to independently and non-invasively visualize MSLN-positive tumors in mouse models with clear imaging contrasts.

These findings demonstrate that despite potential technical challenges due to MSLN shedding, the molecular probe showed remarkable targeting capabilities and is a promising tool for detecting MSLN expression in tumors.

Research Significance and Innovations

This study pioneered the use of single-domain antibodies to develop a PET-based molecular probe, marking the first successful attempt to non-invasively image MSLN using a single-domain antibody fragment. In comparison to traditional molecular probes based on whole antibodies, VHH molecules enable rapid tumor tissue distribution and avoid penetration limitations caused by larger molecular sizes. Thus, the findings of this study are expected to be applied in cancer diagnostics and pre-treatment patient screening and efficacy monitoring in MSLN-related therapies.

Additionally, the probe synthesis process is simple and efficient, with superior PET imaging performance capable of capturing relevant information from tumor tissues in a short period. This provides significant methodological support for future low-cost, high-efficiency tumor monitoring.

Conclusion

The study established the potential and advantages of radiolabeled molecular probes based on single-domain antibodies for cancer diagnostics, addressing the technological gap in imaging MSLN in tumors. These findings not only offer an important tool for cancer clinical practice but also provide an effective method for further exploration of molecular targets in the tumor microenvironment.