"Precision on Two Wheels: Structural Refinement of 68Ga-Labeled Bicyclic Peptides Targeting Nectin-4 for Improved Tumor Imaging: From Preclinical Development to First-in-Human Application"
Today we share a study by Tobias Kronke et al., published in the Journal of Medicinal Chemistry. This research focuses on optimizing the structure of Nectin-4-targeting bicyclic peptides to develop 68Ga-labeled radioligands for tumor imaging. Through systematic structural modifications (such as bioisosteric replacement of methionine), the study not only improved the radiochemical purity and stability of the ligands but also validated their targeting efficacy for the first time in preclinical models and in a first-in-human application, providing a novel molecular tool for the precise diagnosis and treatment of Nectin-4-related cancers.
01 Research Background
Nectin-4 is a cell adhesion protein highly expressed in various tumors (such as urothelial carcinoma) but with limited expression in normal adult tissues (except for embryonic and placental tissues). It has become an important target for antibody-drug conjugates (e.g., enfortumab vedotin) and peptide drugs (e.g., BT8009). In recent years, significant breakthroughs have been made in Nectin-4-targeted therapy:
Clinical Success of Antibody-Drug Conjugates (ADCs): Enfortumab vedotin (Padcev®), the first Nectin-4-targeting ADC, has been approved for the treatment of locally advanced or metastatic urothelial carcinoma, particularly in combination with the PD-1 inhibitor pembrolizumab as a first-line regimen. It employs a cleavable linker to conjugate an anti-Nectin-4 monoclonal antibody with the tubulin inhibitor MMAE, achieving precise cytotoxicity.
Emergence of Peptide-Drug Conjugates (PDCs): BT8009 (zelenectide vedotin) is a novel bicyclic peptide-drug conjugate that uses the same cleavable linker and cytotoxic payload (MMAE) as enfortumab vedotin but utilizes the bicyclic peptide BCY8126 as the targeting module (Figure 1). Preclinical data show excellent antitumor activity, and it is currently in clinical trials for urothelial carcinoma and breast cancer (NCT06225596, NCT06840483).
Figure 1. Structures of BT8009 and BCY8126.
However, a key challenge in current clinical practice is the heterogeneity and dynamic changes in Nectin-4 expression. Kliumper et al. recently found that Nectin-4 expression levels are often significantly reduced or even absent in metastases compared to primary tumors, which is associated with reduced efficacy of enfortumab vedotin. Therefore, non-invasively and accurately quantifying whole-body Nectin-4 expression levels before and during treatment is an urgent clinical need for patient stratification, efficacy prediction, and resistance monitoring. Although antibody-based immunoPET strategies have been explored, the pharmacokinetic properties of large molecules—such as slow blood clearance, poor tumor penetration, and high background—limit their imaging quality and application. In contrast, bicyclic peptides, as an emerging molecular type, offer a perfect balance between size and function:
Moderate Molecular Weight (~2 kDa): Combines good tissue penetration with rapid renal clearance.
High Affinity and Specificity: Selected through techniques like phage display, enabling high-specificity target binding akin to antibodies.
Excellent Stability: The cyclic structure effectively resists protease degradation.
High Modifiability: Amenable to structural optimization and radionuclide conjugation via chemical synthesis.
This study aims to transform the bicyclic peptide targeting head (BCY8126) of the clinical-stage PDC drug BT8009 into an excellent radioligand. The core focus is addressing a key chemical instability issue: the methionine (Met) residue in the BCY8126 sequence is highly susceptible to oxidation to methionine sulfoxide during peptide synthesis and radiolabeling, leading to reduced yield, lower radiochemical purity, and potential impairment of binding activity. To this end, the team systematically explored bioisosteric replacements for methionine, aiming to develop more stable and reliable Nectin-4 PET imaging probes and advance them from preclinical research to first-in-human application.
02 Innovative Highlights
First systematic evaluation of the impact of methionine bioisosteres on bicyclic peptide stability and binding affinity: Innovatively introduced substitutes like norleucine (Nle), methoxinine (Mox), and S-ethylcysteine to address the oxidation risk of methionine (Met) during synthesis and labeling, significantly improving radiochemical purity while maintaining high affinity for Nectin-4.
Multidimensional binding characterization techniques reveal structure-activity relationships: Utilizing fluorescence anisotropy (FA) competitive binding assays and surface plasmon resonance (SPR) kinetic analysis, the study revealed for the first time that replacing 1-naphthylalanine (1NaI) with 2-naphthylalanine (2NaI) led to a 30-fold decrease in affinity, primarily due to a reduced association rate (kon), providing a key basis for subsequent optimization.
Comprehensive validation from preclinical to first-in-human application: Systematically compared the pharmacokinetics of 64Cu/68Ga-labeled ligands from in vitro cell models (HT-1376) to in vivo PET imaging. First reported the successful imaging of [68Ga]Ga-NECT-224 (the optimized ligand based on Mox replacement) in a patient with metastatic urothelial cancer, confirming its clinical translation potential.
03 Results and Discussion
3.1 Peptide Library Design and Binding Affinity Characterization
The study first synthesized a library of bicyclic peptides based on the BCY8126 template (Figure 2), focusing on modifying the Met4 position (e.g., compounds 1e-Met, 3a-Nle, 4-Mox, etc.), and evaluated their binding ability to recombinant human Nectin-4 using FA competition assays. Results showed that the Kd values for Mox (4) and Nle (3a) (1.64 nM and 1.04 nM, respectively) were comparable to the native Met (1e, Kd=0.77 nM), whereas the double substitution 2NaI/Arg5 (8a) led to a significant decrease in affinity (Kd=21.0 nM). SPR kinetics further confirmed that the affinity differences primarily stemmed from changes in the association rate (Figure 3).
Figure 2. Overview of the structures of the bicyclic peptides studied herein.
Figure 3. Binding of peptides to recombinant human nectin-4.
3.2 Radiolabeling and Oxidative Stability Optimization
In 64Cu/68Ga labeling experiments, the Met-containing peptide (1e) was partially oxidized to methionine sulfoxide (2) during the labeling process, leading to the generation of radioactive byproducts (Figure 4). In contrast, the chromatographic peaks for labeled Mox (4) and Nle (3a) were symmetric and single (Figure 4), demonstrating their improved oxidation resistance. H2O2 challenge experiments further showed that the half-life of [64Cu]Cu-4 (Mox) (6.87 h) was significantly higher than that of [64Cu]Cu-1e (Met, 3.5 h) (Figure 5).
Figure 4. Radio-HPLC analysis after 64Cu- and 68Ga-labeling of different peptides.
Figure 5. Time-dependent degradation of selected radiolabeled peptides in the presence of H2O2.
3.3 In Vitro Cell Binding and Internalization Mechanism
In Nectin-4 high-expressing HT-1376 cells, [64Cu]Cu-4 exhibited rapid binding and sustained internalization characteristics: after 60 minutes of incubation, 70% of the ligand was acid-resistant (i.e., internalized), and this process could still occur at 4°C, suggesting a potential non-classical endocytic pathway. Saturation binding experiments determined a Kd of 3.2 nM for [64Cu]Cu-4, consistent with SPR data (Figure 6).
Figure 6. Cell binding and internalization of the Nectin-4-directed radioligands.
3.4 In Vivo PET Imaging and Pharmacokinetics
Animal PET imaging showed that [64Cu]Cu-4 had the highest uptake in HT-1376 tumors (SUVmean peak 1.0), and the tumor-to-muscle ratio was superior to other ligands (Figure 7). Although the initial uptake of [68Ga]Ga-4 was slightly lower, it showed good tumor retention. In the first-in-human application, [68Ga]Ga-NECT-224 successfully detected brain metastases, lymph nodes, and the primary lesion (Figure 8), with a SUVmax reaching 23.8 (renal pelvis tumor), highlighting its clinical value.
Figure 7. PET images and image-derived uptake values of the 64Cu-labeled Nectin-4 ligands.
Figure 8. First-in-human application of [68Ga]Ga-4 ([68Ga]Ga-NECT-224).
04 Conclusion
This study successfully developed novel Nectin-4-targeting bicyclic peptide radioligands through structural optimization. The methoxinine (Mox) replacement strategy significantly enhanced oxidation resistance and radiochemical purity. [64Cu]Cu-NECT-224 and [68Ga]Ga-NECT-224 demonstrated excellent tumor-targeting capability in preclinical models, and the first-in-human application further validated their clinical feasibility. Future work should focus on reducing the dissociation rate (koff) to prolong tumor retention and promoting imaging studies with the longer-half-life 64Cu-labeled version. This study not only advances the development of Nectin-4 imaging probes but also provides a paradigm for the rational design of bicyclic peptides, holding broad translational potential.
Original Article:
Krönke T, Trommer J, Ullrich M, et al. “Precision on Two Wheels”─ Structural Refinement of 64Cu-and 68Ga-Labeled Bicyclic Peptides Targeting Nectin-4 for Improved Tumor Imaging: From Preclinical Development to First-in-Human Application[J]. Journal of Medicinal Chemistry, 2025.