Development of a Mesothelin-Binding Engineered Scaffold Protein as a Theranostic for Pleural Mesothelioma

Our plain-English summary of Development of a Mesothelin-Binding Engineered Scaffold Protein as a Theranostic for Pleural Mesothelioma, by R. Silvestri, M. Piccardi, A. Laurenza, et al (Bioconjugate Chemistry, 2025).

The study

• Authors: R. Silvestri, M. Piccardi, A. Laurenza, F. Rea, A.R. Sirois, M. Lari, F. Bartoli, G. Signore, L. Tedeschi, E. Ferraro, P.A. Erba, R. Giovannoni, S. Landi, F. Gemignani, S.J. Moore
• Journal: Bioconjugate Chemistry
• Published: 2025;36(11):2436-2447
• Cited by: 1 article, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Mesothelin (MSLN) is a tumor biomarker expressed at high levels on the surface of numerous cancers with extremely limited expression in healthy tissues. MSLN-targeting agents developed for diagnosis and therapy could have a significant impact on the management of MSLN-expressing cancers. Pleural mesothelioma (PM) is a deadly cancer that arises from mesothelial cells lining the pleura and is predominantly linked to asbestos exposure. There are currently no effective treatments, and diagnosis occurs in late stages of disease due to the lack of clinical symptoms in the early stages. Recent efforts to diagnose and treat PM have focused on identifying and targeting relevant biomarkers, including MSLN. We engineered proteins based on the nonantibody fibronectin type III (Fn3) protein scaffold that bind MSLN with high affinity and specificity, using yeast-surface display and directed evolution. Previous work with Fn3 scaffold proteins has demonstrated tissue distribution desirable for applications in molecular imaging and targeted radiotherapy, which may overcome limitations encountered thus far with antibody-based approaches to treat PM. The MSLN-targeting Fn3 was further developed for bioconjugation with the 1,4,7,10-tetraazacyclododecane,1-(glutaric acid)-4,7,10-triacetic acid (DOTAGA) radiometal chelator. MSLN-binding Fn3 specifically binds to the MSLN-expressing PM lines, colocalizes with MSLN, and internalizes upon binding. Fn3-DOTAGA was further coupled with cold metal gallium-69, and the resulting conjugate maintained binding with high affinity to MSLN-expressing PM cells. MSLN-binding Fn3-DOTAGA-69Ga is a promising molecule with diagnostic and therapeutic relevance, toward applications in molecular imaging and targeted radiotherapy.

In plain English

This was a laboratory study, not a study of patients. The authors engineered non-antibody fibronectin type III (Fn3) scaffold proteins to bind mesothelin (MSLN), a biomarker on pleural mesothelioma, a cancer the authors describe as predominantly associated with asbestos exposure. Using yeast-surface display and directed evolution, they affinity-matured earlier Fn3 libraries and identified variant 5.3.2 (25% of sequenced variants). Because the MSTO-211H pleural mesothelioma line showed no detectable MSLN, they made MSLN-overexpressing clones; clone 1 and clone 7 gave 12-fold and 10-fold higher staining than wild-type. Fn3 5.3.2 bound clone 1 with KD = 11 ± 4 nM and clone 7 with KD = 11 ± 2 nM, with no binding to wild-type cells; confocal microscopy showed colocalisation with MSLN at 4 °C and internalisation at 37 °C. After adding a cysteine for site-specific conjugation to the DOTAGA chelator, binding was KD = 31 ± 2 nM, and after coupling with cold gallium-69, KD = 15 ± 5 nM. The authors concluded the conjugate is promising for imaging and targeted radiotherapy, with animal work still to come.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory protein-engineering and in vitro cell-binding study (yeast-surface display directed evolution, bioconjugation, flow cytometry and confocal microscopy)
• Who was studied: Engineered Fn3 protein variants tested against the human pleural mesothelioma cell line MSTO-211H (wild-type, MSLN-negative) and two MSLN-overexpressing clones derived from it (MSTO clone 1 and clone 7); work carried out at the University of Pisa, Italy and Smith College, USA
• Exposure: Not an exposure study; the agent tested was MSLN-binding Fn3 variant 5.3.2, its cysteine-modified form conjugated to the DOTAGA chelator, and that conjugate coupled with cold gallium-69
• Main results:
  • Fn3 5.3.2 bound the MSLN-overexpressing mesothelioma clones with KD = 11 ± 4 nM (clone 1) and KD = 11 ± 2 nM (clone 7); no binding was seen to the MSLN-negative wild-type MSTO-211H cells
  • MSLN staining of the engineered clones: clone 1 gave 27-fold higher signal than unlabelled cells and 12-fold higher than wild-type
  • MSLN staining of clone 7: 17-fold higher than unlabelled cells and 10-fold higher than wild-type MSTO-211H
  • At 37 °C Fn3 5.3.2 was internalised into punctate structures in the mesothelioma cells, while the anti-MSLN antibody remained mostly on the membrane
  • The DOTAGA conjugate (Fn3cys-DOTAGA) bound MSLN-expressing cells with KD = 31 ± 2 nM; the gallium-69 coupled conjugate with KD = 15 ± 5 nM
  • Coupling efficiency with gallium-69: 60% with a 10-fold molar excess (15 min, 60 °C, pH 4.5) and 95% with a 100-fold molar excess (15 min, 50 °C, pH 4.5)
  • Thermal stability of Fn3cys-DOTAGA at pH 4.5 without gallium-69: melting temperature about 65 °C
• The authors' conclusion: The authors concluded that Fn3cys-DOTAGA-69Ga was engineered and validated as an MSLN-binding protein with potential for theranostic use in pleural mesothelioma, and that they are now working toward radiolabelled versions and biodistribution testing in animal models
• Limitations the authors state:
  • The authors state that the role of MSLN shedding from the tumour cell surface in imaging and targeted radiotherapy still needs careful assessment in living animals
  • The authors note that cells expressing MSLN at natural levels gave too low a signal for accurate binding measurement, so artificially overexpressing clones had to be made
  • The authors used cold (non-radioactive) gallium-69 as a preliminary stand-in for the radioisotope

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Mesothelin, Pleural Mesothelioma, Biomarker, Mesothelial Cells, Pleura, Asbestos Exposure.

Related studies

• A common polymorphism within MSLN affects miR-611 binding site and soluble mesothelin levels in healthy people — S. Garritano, C. De Santi, et al. (2014)
• Polymorphisms in the putative micro-RNA-binding sites of mesothelin gene are associated with serum levels of mesothelin-related protein — A. Cristaudo, R. Foddis, et al. (2010)
• Decoding the role of mesothelin in tumor dynamics and targeted treatment innovations — R. Silvestri, E. Colucci, et al. (2025)
• Does CA125 binding to mesothelin impact the detection of malignant mesothelioma? — J. Creaney, I.M. Dick, et al. (2013)
• Proceedings of the American Society of Cytopathology companion session at the 2019 United States and Canadian Academy of Pathology Annual meeting, part 2: effusion cytology with focus on theranostics and diagnosis of malignant mesothelioma — M.T. Siddiqui, F. Schmitt, et al. (2019)
• MSLN gene silencing has an anti-malignant effect on cell lines overexpressing mesothelin deriving from malignant pleural mesothelioma — O. Melaiu, J. Stebbing, et al. (2014)

Browse return to medical research.