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Screening, Construction, and Preliminary Evaluation of CLDN18.2-Specific Peptides for Noninvasive Molecular Imaging
Zilei Wang1, Chuanke Zhao2, Jin Ding1
1State Key Laboratory of Holistic Integrative Management of Gastrointestinal Cancers, Beijing Key Laboratory of Carcinogenesis and Translational Research, NMPA Key Laboratory for Research and Evaluation of Radiopharmaceuticals (National Medical Products Administration), Department of Nuclear Medicine, Peking University Cancer Hospital & Institute, Beijing 100142, China.
Abstract:
Recent global clinical trials have shown that CLDN18.2 is an ideal target for the treatment of gastric cancer and that patients with high CLDN18.2 expression can benefit from targeted therapy. Therefore, accurate and comprehensive detection of CLDN18.2 expression is important for patient screening and guidance in anti-CLDN18.2 therapy. Phage display technology was used to screen CLDN18.2-specific peptides from 100 billion libraries. 293TCLDN18.1 cells were used to exclude nonspecific binding and CLDN18.1 binding sequences, while 293TCLDN18.2 cells were used to screen CLDN18.2-specific binding peptides. The monoclonal clones obtained from phage screening were sequenced, and peptides were synthesized based on the sequencing results. Binding specificity and affinity were assessed with a fluorescein isothiocyanate (FITC)-conjugated peptide. A 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated peptide was also synthesized for 68Ga radiolabeling. The in vitro and in vivo stability, partition coefficients, in vivo molecular imaging, and biodistribution were also characterized. Overall, 54 monoclonal clones were selected after phage display screening. Subsequently, based on the cell ELISA results, CLDN18.2 preference monoclonal clones were selected for deoxyribonucleic acid (DNA) sequencing, and four 7-peptide sequences were obtained after sequence comparison; among them, a peptide named T37 was further validated in vitro and in vivo. The T37 peptide specifically recognized CLDN18.2 but not CLDN18.1 and bound strongly to CLDN18.2-positive cell membranes. The 68Ga-DOTA-T37 probe exhibits good in vitro properties and high stability as a hydrophilic probe; it has high biological safety, and positron emission tomography/computed tomography (PET/CT) studies have shown that it can specifically target CLDN18.2 protein and CLDN18.2-positive tumors in mice. 68Ga-DOTA-T37 demonstrated the superiority and feasibility of using a CLDN18.2-specific probe in PCT/CT imaging, which deserves further development and exploitation.
Insights
Researchers developed a novel peptide, T37, using phage display to specifically target CLDN18.2. This peptide, when radiolabeled as 68Ga-DOTA-T37, shows promise for PET/CT imaging of CLDN18.2-positive gastric cancers.
Area of Science:
- Oncology
- Molecular Imaging
- Biotechnology
Background:
- Claudin 18.2 (CLDN18.2) is a validated target for gastric cancer therapy.
- Accurate detection of CLDN18.2 expression is crucial for patient selection in targeted therapy.
Purpose of the Study:
- To develop a CLDN18.2-specific peptide for targeted therapy and molecular imaging.
- To evaluate the specificity, affinity, and in vivo performance of the developed peptide.
Main Methods:
- Phage display technology was employed to screen for CLDN18.2-binding peptides from a large library.
- Peptide specificity was confirmed using cell-based assays with CLDN18.1 and CLDN18.2 expressing cells.
- A selected peptide, T37, was conjugated with DOTA for 68Ga radiolabeling and evaluated in vitro and in vivo.
Main Results:
- Phage display yielded 54 monoclonal clones, with four distinct peptide sequences identified.
- The T37 peptide demonstrated high specificity for CLDN18.2, binding strongly to CLDN18.2-positive cells.
- The 68Ga-DOTA-T37 probe exhibited good in vitro stability, high biological safety, and specific tumor targeting in PET/CT imaging of mice.
Conclusions:
- The T37 peptide is a highly specific CLDN18.2 binder.
- 68Ga-DOTA-T37 is a promising radiotracer for PET/CT imaging of CLDN18.2-positive tumors.
- This CLDN18.2-specific probe warrants further development for clinical applications in gastric cancer.
