Development of a novel molecular probe for visualizing mesothelin on the tumor via positron emission tomography

Abstract

Objectives

Mesothelin (MSLN) is an antigen that is overexpressed in various cancers, and its interaction with tumor-associated cancer antigen 125 plays a multifaceted role in tumor metastasis. The serum MSLN expression level can be detected using enzyme-linked immunosorbent assay; however, non-invasive visualization of its expression at the tumor site is currently lacking. Therefore, the aim of this study was to develop a molecular probe for imaging MSLN expression through positron emission tomography (PET).

Methods

VHH 269-H4 was obtained via immunization of llama using a fragment of MSLN from residue 360 to residue 597. S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) was conjugated to VHH 269-H4 to yield precursor NOTA 269-H4 for radiolabeling. The chelator-to-VHH ratio was determined by mass spectrometry. The binding kinetics of VHH 269-H4 and NOTA 269-H4 were measured by surface plasmon resonance. Flow cytometry was carried out using the anti-mesothelin monoclonal antibody Anetumab to select MSLN-positive and MSLN-negative cell lines. After radiolabeling, the radiochemical purity and in vitro stability were tested by radio-thin-layer chromatography and size exclusion chromatography, respectively. A saturation binding assay was conducted to measure the dissociation constant (K_d) of [⁶⁸Ga]Ga-NOTA-269-H4. By mircoPET/CT imaging and biodistribution studies, the in vivo performances of the novel tracer were investigated in NCG mice bearing OVCAR-8, SKOV-3, or patient-derived xenografts.

Results

VHH 269-H4 targeting MSLN was obtained with a K_d value of 0.3 nM. After conjugation, approximately 27% and 3.2% of VHH were coupled to one and two NOTA chelators, respectively. This yielded precursor NOTA 269-H4 with a K_d value of 1.1 nM. The radiochemistry was accomplished with moderate radiochemical yields (34 ± 14%, n = 9, decay-corrected). [⁶⁸Ga]Ga-NOTA-269-H4 was obtained with high radiochemical purity (> 99%), and was stable after 90 min incubation at room temperature. The binding affinity of the radioligand towards MSLN was kept in the nanomolar range. Flow cytometry revealed that OVCAR-8 cells possess a high level of MSLN expression, while MSLN expression on SKOV-3 cells was negligible. Consistently, in microPET/CT imaging, [⁶⁸Ga]Ga-NOTA-269-H4 demonstrated clear tumor visualization using NCG mice bearing OVCAR-8 xenografts, but no radioactivity accumulation was observed in SKOV-3 xenografts, suggesting a high specificity of the tracer in vivo. In biodistribution studies, [⁶⁸Ga]Ga-NOTA-269-H4 displayed radioactivity accumulation of 2.93 ± 0.39%ID/g in OVCAR-8 xenografts at 30 min post-injection, and the highest tumor-to-blood ratio (~ 3) was achieved at 90 min post-injection. In NCG mice bearing patient-derived xenografts, [⁶⁸Ga]Ga-NOTA-269-H4 was able to noninvasively detect MSLN expression via microPET/CT imaging.

Conclusions

To our knowledge, our studies achieved the first-time to non-invasively detect MSLN expression clearly using a single domain antibody fragment. To sum up, [⁶⁸Ga]Ga-NOTA-269-H4 is a highly promising PET probe to visualize MSLN expression in vivo and holds great potential to monitor MSLN expression during tumor development.