“Preclinical Evaluation of a Trop2-Targeted Peptide Probe for PET Imaging of Triple-Negative Breast Cancer”
Today we are sharing a research article led by Zha Yuan's team, published in Analytical Chemistry. This study, through computationally biology-driven rational design, developed a novel Trop2-targeted peptide probe, [68Ga]Ga-NOTA-PEG2-WP8. It achieved, for the first time, non-invasive and dynamic Positron Emission Tomography (PET) imaging of Trop2 expression in triple-negative breast cancer (TNBC). It was also successfully applied for real-time monitoring of the treatment response to Trop2-targeted Antibody-Drug Conjugates (ADCs), providing a new molecular imaging tool for precision cancer diagnosis and therapy.
01 Research Background
Triple-negative breast cancer (TNBC) is a breast cancer subtype lacking expression of estrogen receptor, progesterone receptor, and HER2, accounting for 15-20% of all breast cancers. It is characterized by high aggressiveness, heterogeneity, and poor prognosis. Current treatment options are limited, primarily relying on chemotherapy, which often leads to drug resistance and recurrence. Trophoblast cell surface antigen 2 (Trop2) is a transmembrane glycoprotein highly expressed in TNBC (44-88.7%). Its overexpression is closely associated with tumor proliferation, invasion, and metastasis, making it an important therapeutic target. In recent years, Trop2-targeted ADCs such as Sacituzumab Govitecan (SG) and Datopotamab Deruxtecan (Dato-DXd) have shown significant efficacy in clinical settings. However, their effectiveness highly depends on Trop2 expression levels, and treatment can lead to Trop2 downregulation or resistance.
Currently, there is a lack of non-invasive tools for dynamically monitoring Trop2 expression, hindering patient stratification and treatment response evaluation. Existing imaging probes are mainly based on antibodies or nanobodies (e.g., 89Zr-DFO-AF650), but their large molecular size results in suboptimal pharmacokinetics (slow clearance, poor penetration), high immunogenicity, and high production costs. Peptide-based probes, owing to their small size, rapid clearance, low toxicity, and high tissue penetration, represent an ideal alternative. This study aimed to design a high-affinity Trop2-targeting peptide via computational simulation and develop a 68Ga-labeled PET probe to address current imaging limitations.
02 Innovative Highlights
Computational Biology-Driven Rational Peptide Design: For the first time, AlphaFold2 molecular docking and FoldX binding free energy calculations were used to simulate the binding interface between Trop2 and its natural ligand Midkine (MDK), identifying key interacting residues (positions 40-47). Through double-saturation mutation optimization, the high-affinity peptide WP8 (binding free energy: -9.3 kcal/mol) was obtained, demonstrating significant improvement over the initial Lead-pep (-8.0 kcal/mol).
Development of the First Small Peptide-Based Trop2 PET Probe: Successful construction of [68Ga]Ga-NOTA-PEG2-WP8, which exhibits nanomolar affinity (SPR-measured KD = 153.3 nM), high radiochemical purity (>98%), and excellent stability (>95% purity in PBS and serum after 120 minutes), filling the gap for peptide-based Trop2 imaging probes.
Breakthrough in Dynamic Therapy Response Monitoring: The probe can sensitively detect Trop2 downregulation induced by Trop2-targeted ADC treatment (SG and Dato-DXd). Through in vitro cell experiments, small animal PET imaging, and ex vivo validation, it enables non-invasive, real-time visualization of treatment response, providing a new tool for predicting ADC efficacy.
03 Results and Discussion
3.1 Computational Simulation and Peptide Optimization
The binding interface between MDK and Trop2 (PDB: 7PEE) was simulated using AlphaFold2, identifying the key peptide segment Lead-pep . FoldX was used for single-point and double-saturation mutation scanning, revealing that substituting Ala41 and Cys45 with Tyr significantly enhanced affinity, ultimately yielding the optimized peptide WP8 . Molecular dynamics simulations showed that the WP8-Trop2 complex had lower RMSD fluctuations (3.5-4.5 Å), indicating more stable binding . Surface Plasmon Resonance (SPR) confirmed the KD of NOTA-PEG2-WP8 to be 153.3 nM (Fig. 1).
Figure 1. Virtual screening and in vitro validation of candidate peptides.
3.2 Probe Preparation and Characterization
After solid-phase synthesis and 68Ga labeling, [68Ga]Ga-NOTA-PEG2-WP8 exhibited a radiochemical purity >98% (HPLC analysis). After 120 minutes of incubation in PBS and fetal bovine serum (FBS), purity remained >95%, demonstrating excellent stability. The log P value was -2.32 ± 0.12, indicating high hydrophilicity conducive to rapid renal clearance. In vivo blood stability experiments showed a high proportion of intact probe in the blood 60 minutes post-injection (Fig. 2B).
Figure 2. Chemical structure and quality control of [68Ga]Ga-NOTA-PEG2-WP8.
3.3 In Vitro Specificity and Affinity Assessment
Western blot and immunofluorescence confirmed significantly higher Trop2 protein levels in MDA-MB-468 cells (Trop2-high) compared to MDA-MB-231 cells (Trop2-low). Time-dependent uptake experiments showed that [68Ga]Ga-NOTA-PEG2-WP8 uptake in MDA-MB-468 cells peaked at 2.58 ± 0.13% AD (120 minutes), and this uptake could be blocked by an excess of unlabeled peptide (reduced to 0.81 ± 0.06% AD). Competitive binding assays yielded an IC50 of 91.22 nM, confirming high affinity and specificity (Fig. 3).
Figure 3. In vitro specificity assay of [68Ga]Ga-NOTA-PEG2-WP8.
3.4 In Vivo PET Imaging and Pharmacokinetics
Small animal PET imaging revealed significantly higher probe uptake in Trop2-high MDA-MB-468 tumors (peak: 4.09 ± 0.38% ID/g) compared to low-expressing MDA-MB-231 tumors (2.50 ± 0.43% ID/g). Blocking experiments confirmed uptake specificity (decreased to 2.00 ± 0.28% ID/g). The tumor-to-muscle (T/M) ratio reached 4.93 ± 0.57 at 60 minutes, demonstrating a good target-to-background ratio. Pharmacokinetic analysis indicated the probe was primarily cleared renally (kidney uptake: 14.97 ± 3.26% ID/g) with rapid blood clearance (distribution half-life: 1.67 minutes) (Fig. 4).
Figure 4. MicroPET imaging of [68Ga]Ga-NOTA-PEG2-WP8 in MDA-MB-468 and MDA-MB-231 tumor-bearing mice.
3.5 ADC Treatment Response Monitoring
After treating MDA-MB-468 cells with SG and Dato-DXd, Western blot and immunofluorescence showed dose-dependent downregulation of Trop2 expression. Cell uptake experiments confirmed the probe's ability to sensitively detect these expression changes. In animal models, ADC treatment groups (SG 10 mg/kg or Dato-DXd 10 mg/kg) showed significant tumor volume inhibition. PET imaging revealed decreasing probe uptake over the treatment course: the T/M ratio in the SG group decreased from a baseline of 5.23 ± 0.34 to 1.55 ± 0.37 at day 18. Ex vivo autoradiography, Western blot, and immunofluorescence all verified Trop2 downregulation (Fig. 5).
Figure 5. Therapy monitoring of MDA-MB-468 tumor-bearing mice treated with PBS, SG, and Dato-DXd.
3.6 Biodistribution and Toxicity Assessment
Biodistribution studies showed the probe mainly accumulated in the kidneys and bladder, indicating urinary excretion, with low uptake in other organs. H&E staining confirmed that ADC treatment caused no significant toxicity to major organs, while tumor tissue structure was disrupted, indicating treatment safety.
04 Conclusion and Future Perspectives
This study successfully developed the first Trop2-targeted peptide PET probe, [68Ga]Ga-NOTA-PEG2-WP8. Through rational design aided by computational modeling, the high-affinity peptide WP8 was obtained. The probe exhibits excellent specificity, stability, and pharmacokinetic properties. In preclinical models, it enabled non-invasive, dynamic monitoring of Trop2 expression and ADC treatment response, providing a powerful tool for precise stratification, efficacy evaluation, and resistance management in TNBC. Future work will focus on advancing clinical translation studies to validate its utility in patients and exploring its potential in other Trop2-positive cancers (e.g., lung cancer, pancreatic cancer). Furthermore, probe structure could be optimized to enhance tumor retention time, and versions labeled with therapeutic radionuclides (e.g., 177Lu) could be developed for targeted radionuclide therapy.
Original Article:
Zha Y, Zhu X, Xue Y, Huang Z, Wang S, Jiao Y, Chen Y, Yao Y, Wang K, Fang J. Preclinical Evaluation of a Trop2-Targeted Peptide Probe for PET Imaging of Triple-Negative Breast Cancer. Anal Chem. 2025 Oct 28;97(42):23598-23608. doi:10.1021/acs.analchem.5c05242. Epub 2025 Oct 17. PMID: 41105928.