Preliminary biological evaluation of a [¹⁷⁷Lu]Lu-labeled peptide targeting Nectin-4

Abstract

Background

Despite the clinical implementation of Nectin-4-targeted antibody-drug conjugates (ADCs) and radioligand therapies (RLTs), their therapeutic window is often restricted by systemic toxicities and the emergence of drug resistance. To circumvent these limitations, we developed [¹⁷⁷Lu]Lu-DOTA-AC-SP, a novel peptide-based radiopharmaceutical specifically engineered for the targeted treatment of Nectin-4-expressing malignancies.

Results

The synthesis of [¹⁷⁷Lu]Lu-DOTA-AC-SP was achieved with high radiochemical purity and excellent stability in vitro. Pharmacokinetic evaluations revealed a favorable profile characterized by high binding affinity and rapid systemic clearance. In vivo biodistribution studies demonstrated significant tumor accumulation and prolonged retention, resulting in superior target-to-background ratios. Furthermore, [¹⁷⁷Lu]Lu-DOTA-AC-SP exhibited robust anti-tumor efficacy in Nectin-4-positive models, maintaining an excellent safety profile with minimal off-target effects.

Conclusions

These findings characterize [¹⁷⁷Lu]Lu-DOTA-AC-SP as a potent and safe therapeutic candidate for Nectin-4-positive tumors. Its favorable pharmacokinetic properties and significant therapeutic index support its potential clinical translation as a next-generation treatment for urothelial carcinoma (UC).

Supplementary Information

The online version contains supplementary material available at 10.1186/s41181-026-00435-1.

Introduction

Urothelial carcinoma (UC) is the most common malignant tumor of the urinary system(Antoni et al. 2017). Globally, approximately 516,000 new UC cases were diagnosed in 2020(Richters et al. 2020; Sung et al. 2021), and this number is projected to increase to 586,000 and 663,000 by 2025 and 2030, respectively. Because UC is basically asymptomatic in the early stage, about 45% of patients are diagnosed at an advanced stage, and 10%–15% already present with metastatic disease, severely compromising their quality of life and prognosis )(Galsky et al. 2011; Lenis et al. 2020). Therefore, the development of therapeutic drugs for UC is of great necessity.

Nectin cell adhesion molecule 4(Nectin-4) is a type I transmembrane glycoprotein belonging to the Nectin family of cell adhesion molecules. It plays a pivotal role in the formation and maintenance of adherens junctions by interacting with other Nectins or cadherins. Under physiological conditions, Nectin-4 expression is predominantly limited to embryonic development and is weakly expressed in adult healthy tissues. However, it is significantly overexpressed in various epithelial malignancies, particularly in UC, where it promotes tumor cell proliferation, migration, and angiogenesis through the activation of the PI3K/Akt signaling pathway. (DeRycke et al. 2010; Heath and Rosenberg 2021; Takano et al. 2009). Based on this, in 2019, Enfortumab vedotin (EV), a Nectin-4-targeting ADC, received FDA approval for treating locally advanced and metastatic UC. Numerous clinical trials, including EV-101 and EV-302, have confirmed the promising clinical activity of EV for UC, with improved survival outcomes even in those with poor prognostic factors, such as liver metastases(Hoimes et al. 2023; Powles et al. 2021; Yu et al. 2021)^,. However, EV therapy, especially when combined with pembrolizumab, also has non-negligible concerns: a high incidence of adverse events such as skin toxicity (67.1%) and neuropathy (60.5%), and, as an ADC therapy, the development of acquired resistance is also inevitable ^,(Klümper et al. 2023; Rosenberg et al. 2019). Thus, there remains a strong need for the further development of therapeutic drugs for UC.

Radiopharmaceuticals carrying therapeutic medical isotopes can achieve targeted killing of tumors through internal radiation. Compared with traditional chemotherapeutic agents, radiopharmaceuticals offer the advantages of fewer side effects and lower drug resistance. In addition, the “bystander effect” enables them to achieve better therapeutic efficacy. For Nectin-4-targeting therapeutic drugs, there have also been reports of radionuclide-conjugated drugs (RDC) using antibodies as the targeting molecule, labeled with ¹⁷⁷Lu (Sathekge et al., n.d.) and ²²⁵Ac (Babeker et al. 2025). However, the use of antibodies as targeting molecules comes with significant limitations due to their molecular weight, such as poor tumor penetration and slow blood clearance, which can lead to high background radiation and hematotoxicity(Larson et al. 2015). In addition, antibody-based drugs also carry the risk of inducing immunogenicity. In comparison, due to faster tissue distribution and excellent tumor penetration, as well as specific targeting ability, peptide-based radiopharmaceuticals have achieved remarkable clinical and commercial success, such as Pluvicto([¹⁷⁷Lu]Lu-PSMA-617 targeting PSMA) used for castration-resistant prostate cancer(van der Sar et al. 2022) and Lutathera([¹⁷⁷Lu]Lu-DOTATATE targeting SSTR) used for neuroendocrine tumors(Fortunati et al. 2022).In recent years, there have been several reports on peptide-drug conjugate, especially bicyclic peptide, for Nectin-4(Baldini et al. 2025; Mudd et al. 2022). In addition, some peptide-based radiodiagnostic agents, such as[⁶⁸Ga]Ga-NOTA-SP(Li et al. 2025), have also been developed. To explore the feasibility of developing peptide-based radiotherapeutic drugs, a novel simple-structured monocyclic peptide radioligand DOTA-AC-SP was designed based on BT8009, which is a bicyclic peptide-drug conjugate with superior Nectin-4 affinity and pharmacokinetic properties. The monocyclic peptide retains the Nectin-4 binding sequence of the bicyclic peptide, and its monocyclic scaffold also offers lower synthesis costs. After being radiolabeled with ¹⁷⁷Lu, the preclinical biological experiments, including binding affinity, cellular uptake, biodistribution, and therapeutic efficacy in Nectin-4-positive tumor models, were conducted to evaluate its clinical application potential.

Materials and methods

Materials

The peptide DOTA-AC-SP was commissioned to be synthesized by Sentinel (Shanghai, China) (Suppl. Figure 1–2):.[¹⁷⁷Lu]LuCl₃ solution was obtained from ITM Isotopen Technologien München AG. Antibody against Nectin-4was purchased from Proteintech Group, Inc. The cell lines SW780, MDA-MB-231, and the mouse model were purchased from Shanghai Chuanqiu Biotechnology Co., Ltd. and Serena Life Science Technology Development (Shanghai) Co., Ltd. DMEM medium containing 10% FBS and glutamine was purchased from Gibco, USA. PBS and DAB were purchased from Thermo Fisher Scientific (China) Co., Ltd. The other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

Molecular docking and DOTA-AC-SP cell flow cytometry

Molecular docking simulations were performed to investigate the binding mode of DOTA-AC-SP with Nectin-4. The 3-dimensional crystal structure of Nectin-4 was retrieved from the Protein Data Bank(PDB), while the structure of DOTA-AC-SP was modeled using ChemDraw and subsequently energy-minimized. Both the receptor and ligand were pre-processed (including the addition of polar hydrogens and assignment of Gasteiger charges) using AutoDock Tools. The docking process was executed via AutoDock Vina. The resulting binding poses and intermolecular interactions were visualized and analyzed using PyMOL software.

The binding affinity of DOTA-AC-SP for Nectin-4 was further assessed by flow cytometry using SW780 (high Nectin-4 expression) and MDA-MB-231 (moderate Nectin-4 expression) cells. SW780 cells were assigned to groups A and C, and MDA-MB-231 cells to group B. Both of the cells were seeded in 12-well plates at a density of 2 × 10⁵ cells per well and allowed to adhere overnight. Then, the medium was removed, and group A (SW780) was treated with PBS as a negative control, while groups B (MDA-MB-231) and C (SW780) were incubated with FITC-conjugated DOTA-AC-SP at a concentration of 10 µg/mL. After a 2-hour incubation at 4 °C in the dark, cells were washed twice with cold PBS, resuspended in 300 µL of PBS, and analyzed on a flow cytometer. Fluorescence intensity was measured using the FITC channel (excitation 488 nm, emission 530 nm), with data from at least 10,000 events collected per sample.

Preparation of [¹⁷⁷Lu]Lu-DOTA-AC-SP

[¹⁷⁷Lu]Lu-DOTA-AC-SP was prepared via a one-step radiolabeling procedure. Briefly, 10 µL of peptide solution (1 µg/µL dissolved in 0.5 M sodium acetate buffer, pH 5.20) was added to the reaction tube, followed by the addition of 2 µL of [¹⁷⁷Lu]LuCl₃ solution (37 MBq in 0.04 M HCl). The reaction mixture was incubated at 60.0 °C for 15 min. After the reaction, the mixture was diluted with 188 µL of sodium acetate buffer. Then, the labeling efficiency was determined using both radio-thin layer chromatography (radio-TLC) and radio-high performance liquid chromatography (radio-HPLC) For radio-TLC analysis, iTLC-SG strips were employed as the stationary phase, and a mixture of saline and 0.05 M hydrochloric acid (100:0.5, v/v) was used as the mobile phase. Under this condition, [¹⁷⁷Lu]Lu-DOTA-AC-SP exhibits an R_f value of 0–0.02, while [¹⁷⁷Lu]Lu³⁺ migrates with an R_f of 0.86–1.0. Radio-HPLC analysis was performed using a Kromasil 100-5 C18 column (250 × 4.6 mm, 5 μm) with mobile phase A (H₂O containing 0.1% trifluoroacetic acid) and mobile phase B (acetonitrile containing 0.1% trifluoroacetic acid). The chromatograms for the preliminary cold-labeled Lu-DOTA-AC-SP and the final labeled compound are shown in Suppl. Figure 3 A.B.

In vitro stability of [¹⁷⁷Lu]Lu-DOTA-AC-SP

To evaluate the in vitro stability of [¹⁷⁷Lu]Lu-DOTA-AC-SP, 20µL of [¹⁷⁷Lu]Lu-DOTA-AC-SP was incubated with 200 µL of phosphate-buffered saline (PBS, pH 7.4). The mixtures were maintained at 37 °C and sampled at different time points. Then they were analyzed by radio-TLC to determine the radiochemical purity.

In vitro biological studies

Cellular experiments

Uptake experiments

Cellular uptake assays were conducted using the SW780 cell line. Briefly, cells were seeded uniformly in 24-well plates at a density of 10⁵ cells per well and cultured for 48 h. Before the experiment, the culture medium was aspirated, and the cells were gently washed twice with fresh culture medium. Then, the cells were incubated with 37 kBq of [¹⁷⁷Lu]Lu-DOTA-AC-SP in serum-free medium at 37 °C for different time periods (30, 60, 90, 120, or 180 min). To assess binding specificity, a blocking group was also included, in which the cells were pre-incubated with unlabeled DOTA-AC-SP (5 µg/well) for 15 min, followed by the administration of [¹⁷⁷Lu]Lu-DOTA-AC-SP. At the end of each incubation period, the supernatant was removed and collected. The cells were then washed twice with ice-cold PBS and subsequently lysed with 0.5 mL of 0.5 M NaOH. The resulting PBS and lysates were collected, and the associated radioactivity was quantified using an automatic γ-counter, as well as the supernatant. All experiments were performed with five replicate wells and one blank well. Cellular uptake was calculated as the percentage of the incubated dose taken up per 10⁵ cells (%ID/10⁵ cells) according to the following formula, where the measured values were corrected by subtracting the blank well value:

Saturation binding assay

The cells (10⁵ cells/mL) were added to 24-well plates (2 mL/well) for saturation binding assays. [¹⁷⁷Lu]Lu-DOTA-AC-SP (15–20 GBq/µmol) with activities of 1.85, 3.7, 7.2, 18.5, 37, and 72 kBq was added to each well and then incubated for 4 h in 37℃. To determine non-specific binding (NSB), a parallel set of wells was incubated with unlabeled DOTA-AC-SP (5 µg/well). After washing and lysis, the radioactivity counts in the lysate were determined, and the specific binding was calculated by subtracting the NSB from the total binding at each concentration. Subsequently, the radioactivity counts were converted into molar amounts (nmol). Then, the specific binding and the concentration of [¹⁷⁷Lu]Lu-DOTA-AC-SP were summarized and fitted to a curve. The reciprocal of the slope of the fitted curve obtained by Scatchard analysis represents the K_d value, and the intercept of the fitted curve on the x-axis is the B_max value.

Plasma concentration-time curves

All animal experiments were conducted in accordance with the institutional guidelines and approved by the Animal Ethics Committee under protocol number Vista-IA-2-1-2306-01. Normal BALB/c mice (n = 6; 3 males and 3 females), 6–8 weeks old, were used to determine the plasma pharmacokinetic profile of [¹⁷⁷Lu]Lu-DOTA-AC-SP. Each mouse received an intravenous injection of 100µL of [¹⁷⁷Lu]Lu-DOTA-AC-SP (2.8–3.7 MBq, diluted with PBS, 15–20 GBq/µmol) via the tail vein. Blood samples were collected at designated time points by tail vein puncture using capillary tubes of known mass. The samples were transferred into pre-weighed counting tubes, and the radioactivity was measured using an automatic γ-counter. The mass of each blood sample was determined by the gravimetric method. The percentage of injected dose per gram of tissue (%ID/g) was calculated using the following formula:

Plasma concentration-time (%ID/g-time) curves were plotted using Origin software, and pharmacokinetic parameters were calculated after curve fitting using DAS 2.0 software with a non-compartmental model.

Small animal SPECT/CT imaging

A suspension of SW780 cells (5 × 10⁶ cells in 0.1 mL) was mixed with Matrigel matrix gel at a 2:1 ratio (v/v). The mixture was then injected subcutaneously into the flank of male BALB/c mice to establish xenograft tumors, which were allowed to grow until they reached a volume of 100–500 mm³ before being used for imaging studies. SPECT/CT imaging was performed at 4, 24, and 36 h post-intravenous injection of 3.7 MBq [¹⁷⁷Lu]Lu-DOTA-AC-SP. Regions of interest (ROIs) were delineated around the tumors and major organs.

Therapeutic efficacy

Thirty BALB/c mice (male) bearing 100–500 mm³ of SW780 xenograft tumors were randomly assigned to six experimental groups (n = 5 per group): high-dose (18.5 MBq per mouse), medium-dose (7.4 MBq per mouse), low-dose (3.7 MBq per mouse), [¹⁷⁷Lu]Lu-only control (7.4 MBq per mouse of [¹⁷⁷Lu] LuCl₃), and saline control. The body weight was measured, and the tumor size was calculated using the formula: Volume = (longest diameter × shortest diameter²)/2 every 48 h following administration. Survival analysis was performed based on predefined humane endpoint criteria.

Terminal biodistribution

Upon completion of the therapeutic efficacy study, mice were euthanized and subjected to systematic dissection. Tumors and major organs—including the heart, liver, spleen, lungs, kidneys, stomach, small intestine, muscle, brain, and blood—were collected and weighed. All tissue samples were processed following a standardized dissection protocol. After gravimetric measurement, the radioactivity in each sample was quantified using an automatic γ-counter. The percentage of injected dose per gram of tissue (%ID/g) was calculated after decay correction using the following formula:

Histopathological and hematological analyses

At the predefined therapeutic endpoint, major organs—including the heart, liver, spleen, lungs, and kidneys—were harvested for histological examination using hematoxylin and eosin (H&E) staining. Concurrently, tumor tissues were collected for immunohistochemical (IHC) analysis. Whole blood samples were also obtained for biochemical assessment, which included evaluating hepatic function through alanine aminotransferase (ALT) levels and renal function via blood urea nitrogen (BUN) measurements.

Blood sample processing

Whole blood aliquots underwent 30-minute coagulation at ambient temperature, followed by centrifugation (3,000 rpm × 15 min) to isolate serum for subsequent assays.

Hematoxylin-eosin staining and immunohistochemistry

The collected tumor and other tissues were fixed in a fixative solution for over 24 h. Then, they were dehydrated through a graded ethanol series, embedded in paraffin, and sectioned.

For histological evaluation, sections were stained with hematoxylin and eosin (H&E). After staining, the slides were dehydrated in absolute ethanol and xylene sequentially before being sealed with coverslips. Subsequently, microscopic examination was performed, followed by image acquisition and analysis.

For IHC staining, sections were subjected to antigen retrieval using Tris-EDTA buffer (pH 9.0) in a pressure cooker for 5 min after boiling was achieved, followed by natural cooling to room temperature. After three 5-minute washes with PBS, the sections were permeabilized with 0.2% Triton X-100 for 25 min at room temperature and again washed three times with PBS. Endogenous peroxidases were blocked by incubation with 3% hydrogen peroxide for 10 min at room temperature, followed by another three PBS washes. The sections were then incubated with 1× animal-free blocking solution for 10 min at room temperature. Then, the excess blocking solution was removed, and the sections were incubated overnight at 4 °C with a primary antibody against Nectin-4. After warming to room temperature, the sections were washed three times with PBS and incubated with a goat anti-rabbit secondary antibody for 1 h at room temperature. Following three additional PBS washes, antigen visualization was performed using DAB substrate for 5 min, and counterstaining was carried out with hematoxylin for 3 min. Finally, the sections were dehydrated through an ascending alcohol series (90% and 100% ethanol, 3 min each) and cleared in xylene (two changes, 5 min each). Mounting was performed using neutral balsam, and images were acquired under a microscope for subsequent analysis.

Statistical analyses

The final results of the independent parallel experiments were processed using Excel software and expressed as mean ± standard deviation (SD).

Results

Molecular docking and DOTA-AC-SP cell flow cytometry

Molecular docking simulations were performed to characterize the molecular interactions between DOTA-AC-SP and the Nectin-4 receptor. The results indicated a stable binding conformation between the peptide and the receptor, with a calculated binding energy of − 6.6 kcal/mol, primarily mediated by specific contacts with key residues such as ARG-115 and GLN-110 (Fig. 1A). To validate these computational findings, flow cytometry was carried out. As shown in Fig. 1B, after treated with FITC-labeled DOTA-AC-SP, the SW780 cells exhibited a marked increase in fluorescence signal relative to both the control group and group MDA-MB-231 cells, further confirming the favorable binding specificity of the peptide for Nectin-4.

Fig. 1.

A Molecular docking of DOTA-AC-SP to Nectin-4. B Flow cytometry histogram of SW780 and MDA-MB-231cells after incubation with PBS or 0.005 µg/µL of FITC-AC-SP. (Fluorescein isothiocyanate isomer)

Preparation and in vitro stability of [¹⁷⁷Lu]Lu-DOTA-AC-SP

As shown in Fig. 2A, to obtain the optimized radiolabeling conditions, the effects of temperature, reaction time, and pH on labeling efficiency were systematically investigated. In each labeling reaction, approximately 5–10 µg of the conjugate (DOTA-AC-SP) was utilized. The results indicate that the labeling temperature exerts a significant impact on the labeling efficiency, which tends to decrease due to the radiolysis of the ligand and labeled product at relatively high temperatures. Regarding reaction time, because of the relatively low reaction temperature, a minimum of 15 min was required to achieve sufficient complexation of [¹⁷⁷Lu]Lu with the ligand. Within the investigated range, the labeling efficiency of [¹⁷⁷Lu]Lu-DOTA-AC-SP is relatively high under high pH(5.20). Under optimized conditions (Fig. 2B, the reaction temperature, time, and pH were 60 °C, 15 min and 5.20, respectively), the labeling efficiency of [¹⁷⁷Lu]Lu-DOTA-AC-SP consistently exceeded 95% (Fig. 2C), and it was relatively stable in PBS, with a radiochemical purity of greater than 90% at 6 days (Fig. 2D).

Fig. 2.

A The effect of reaction temperature (a), time (b), and pH(C) on the labeling efficiency of [¹⁷⁷Lu]Lu-DOTA-AC-SP (Except for the variable parameters, the reaction temperature, time, and pH were 60 °C, 15 min, 5.20, respectively); B The preparation of [¹⁷⁷Lu]Lu-DOTA-AC-SP under optimized conditions; C HPLC chromatogram of [¹⁷⁷Lu]Lu-DOTA-AC-SP; D In vitro stability of [¹⁷⁷Lu]Lu-DOTA-AC-SP(Radiochemical purity determined by TLC)

In vitro cellular experiments

Due to the high expression of Nectin-4 in the SW780 cell line, which is derived from human urothelial carcinoma, the cellular uptake of [¹⁷⁷Lu]Lu-DOTA-AC-SP can reach 0.95%ID/10⁵ cells within 0.5 h in the experimental group. By comparison, because of the competitive effect with excess DOTA-AC-SP, the cell uptake of [¹⁷⁷Lu]Lu-DOTA-AC-SP in the blocking group was only 0.5%ID/10⁵cells at 0.5 h, and also significantly lower than that in the experimental group at other time points (Fig. 3A). It was also found that the cell uptake in both experimental and blocking groups decreased with the increase of time after 0.5 h, which might be due to the cytotoxic effect of ¹⁷⁷Lu on cells. Nevertheless, saturation binding assays still confirmed that [¹⁷⁷Lu]Lu-DOTA-AC-SP binds to SW780 cells with nanomolar affinity, yielding a dissociation constant (K_d) of 2.08 nM and a maximum binding capacity (B_max) of 2.35 fmol/10⁵cells (Fig. 3B).

Fig. 3.

A Cellular uptake of experimental and blocking groups at different times (n = 5). B Saturation binding constants and maximum binding of [¹⁷⁷Lu]Lu-DOTA-AC-SP to SW780 cells (n = 5)

Plasma concentration-time curves

The plasma concentration-time profile of [¹⁷⁷Lu]Lu-DOTA-AC-SP, expressed as %ID/g versus time, is presented in Fig. 4. Following intravenous administration, [¹⁷⁷Lu]Lu-DOTA-AC-SP exhibited restricted tissue distribution and rapid blood clearance. The maximum plasma concentration was observed at the first sampling time point (2 min), after which the concentration declined rapidly over time due to non-specific cellular uptake, hydrolysis in circulation, and specific binding to target tissues. The terminal elimination half-life (t₁/₂) of [¹⁷⁷Lu]Lu-DOTA-AC-SP was calculated to be 83.6 min, which is consistent with the characteristic rapid pharmacokinetic profile of peptides.

Fig. 4.

Plasma concentration-time curve of [¹⁷⁷Lu]Lu-DOTA-AC-SP (n = 6)

SPECT/CT imaging of [¹⁷⁷Lu]Lu-DOTA-AC-SP

To evaluate the biodistribution and tumor targeting of [¹⁷⁷Lu]Lu-DOTA-AC-SP in SW780 tumor-bearing mice, the small-animal SPECT/CT imaging was performed. As shown in Fig. 5A, tumor uptake of [¹⁷⁷Lu]Lu-DOTA-AC-SP was observable as early as 4 h post-injection and retained in the tumor for approximately 36 h. Significant accumulation was also noted in the kidneys and bladder, consistent with the typical metabolic and excretory pathway of peptide-based radiotracers, which involves proteolytic degradation.

Fig. 5.

A Small animal SPECT-CT imaging of tumor-bearing mice injected with [¹⁷⁷Lu]Lu-DOTA-AC-SP. B Tumor-to-non-tumor ratios (n = 3)

The tumor-to-non-tumor (T/NT) ratios are summarized in Fig. 5B. Specific retention of [¹⁷⁷Lu]Lu-DOTA-AC-SP in tumor tissue resulted in gradually increasing T/NT ratios over time, with tumor-to-muscle (T/M) ratios reaching 5.90 ± 0.85, 7.89 ± 1.23, and 8.84 ± 1.62 at 4, 24, and 36 h. Together, these SPECT/CT imaging results demonstrate that [¹⁷⁷Lu]Lu-DOTA-AC-SP exhibits significant accumulation in Nectin-4-positive tumors with high contrast relative to muscle and bone tissues, which preliminarily support the potential of [¹⁷⁷Lu]Lu-DOTA-AC-SP as a radiotherapeutic agent for Nectin-4-expressing malignancies.

Therapeutic efficacy

The therapeutic potential of [¹⁷⁷Lu]Lu-DOTA-AC-SP was evaluated in SW780 tumor-bearing mice. Throughout the experimental period, rapid tumor growth was observed in the saline-treated control group, where the tumor volume reached 812.5 ± 57.9 mm³, with an increase factor of 4.00. In contrast, tumor growth was significantly slower in the high, medium, and low-dose treatment groups, with final average volumes of 170.1 ± 22.8 mm³, 299.0 ± 49.1 mm³, and 408.4 ± 46.8 mm³, corresponding to only 1.14, 1.26, and 2.03-fold increases to baseline, respectively (Fig. 6A). Furthermore, treatment with [¹⁷⁷Lu]Lu-DOTA-AC-SP significantly prolonged the survival of SW780 tumor-bearing mice. All animals in the medium-dose group survived until the end of the treatment period, whereas mice treated with saline or [¹⁷⁷Lu]Lu alone exhibited substantial mortality during the study (Fig. 6B). No significant body weight loss was observed in any group, preliminarily indicating a favorable safety profile without systemic toxicity of [¹⁷⁷Lu]Lu-DOTA-AC-SP, which can further support its therapeutic efficacy (Fig. 6C).

Fig. 6.

A Tumor volume, B Mouse weight changes, and C Survival rate in therapeutic experiments (n = 5); D Biodistribution in the major organs of tumor-bearing mice at the end of treatment (n = 3)

Terminal biodistribution

At day 14 post-administration, tumor-bearing mice were euthanized and dissected for biodistribution analysis. As shown in Fig. 6D, significant radioactive retention in tumor tissues was observed in all administration groups with different doses. Since [¹⁷⁷Lu]Lu-DOTA-AC-SP is mainly metabolized by the liver and kidneys, its accumulation in these two organs exhibits a significant dose-dependent pattern. In contrast, the distribution in other organs is mainly limited by blood flow or tissue affinity rather than metabolic rate, and thus, within a certain dose range, the distribution in these tissues does not change significantly. (The source data for the Terminal Biodistribution are presented in Tables 1, 2, 3 and 4 of the Supporting Information.)

HE staining after treatment

After the treatment concluded, HE staining was performed on major organs of SW780 tumor-bearing mice to further evaluate the potential toxicity and therapeutic effects of [¹⁷⁷Lu]Lu-DOTA-AC-SP. In comparison with the saline group, there were no significant pathological changes in the heart, liver, spleen, lungs, or kidneys in the other groups. Notably, significant necrotic areas were observed in the tumors of mice administered high, medium, and low doses of [¹⁷⁷Lu]Lu-DOTA-AC-SP, while minor necrosis was also detected in the [¹⁷⁷Lu]Lu groups. In contrast, tumors treated with saline exhibited well-preserved morphological structure with no necrotic regions (Fig. 7A).

Fig. 7.

A Histological features of HE-stained tissues, B Nectin-4 expression levels in tumors and C ALT, Urea levels in various groups after treatment(Scale bar represents 500 μm for all panels)

Immunohistochemical analysis

Immunohistochemical results demonstrated that the Nectin-4 expression in the tumor was strongest in the saline group and slightly reduced in the group treated with [¹⁷⁷Lu]Lu alone. Remarkably, a pronounced decrease in Nectin-4 expression was observed in the tumors of the low, medium, and high-dose groups, correlating with increasing doses of [¹⁷⁷Lu]Lu-DOTA-AC-SP. This result confirms that [¹⁷⁷Lu]Lu-DOTA-AC-SP can specifically bind to tumor cells via the targeting affinity of DOTA-AC-SP and exert potent tumoricidal effects through the β-radiation emitted by [¹⁷⁷Lu]Lu, which consequently leads to a significant reduction in Nectin-4 expression levels in tumor tissues(Fig. 7B).

Hematological analysis

After the completion of treatment, the ALT and UREA levels in the high-, medium-, and low-dose groups were all within the acceptable ranges in the blood biochemical analysis. Furthermore, there is no significant difference in ALT and UREA values between any of the groups and the saline group (Fig. 7C), initially indicating that the use of [¹⁷⁷Lu]Lu-DOTA-AC-SP has no impact on liver and kidney functions.

Discussion

UC remains a significant global public health challenge due to its occult nature and high mortality rate, thus, the development of innovative treatment strategies is meaningful to improving patient survival and therapeutic outcomes. The marketing of ADC EV has proven that Nectin-4 is an effective target for the treatment of UC. However, the issues of side effects and drug resistance still need to be further addressed.

Given the unique advantages that radiopharmaceuticals offer for precision medicine, a growing number of Nectin-4-targeting radiotracers have been extensively explored for screening of tumor patients and real-time monitoring of therapeutic responses, including antibody-based agents such as ^99mTc-HYNIC-mAb and ⁸⁹Zr-AGS-22M6(Shao et al. 2022)^,(Campbell et al. 2016), as well as peptide-based probes such as ⁶⁸Ga-FZ-NR-1, ⁶⁸Ga-FZ-NR-2, and ⁶⁸Ga-N188(Sun et al. 2025)⁻(Zhang et al. 2024). In addition, there have also been several reports on Nectin-4-targeted radiotherapeutic drugs using full antibodies as vectors. However, the use of antibodies as vectors for the preparation of radiopharmaceuticals carries potential hematological toxicity. Furthermore, linking therapeutic radionuclides to vectors originally designed for diagnostic radiopharmaceuticals may suffer from insufficient tumor retention. Therefore, it is still necessary to further develop peptide-based Nectin-4-targeted therapeutic radiopharmaceuticals.

Based on the above, we developed a [¹⁷⁷Lu]Lu-labeled Nectin-4-targeting peptide RDC, aiming to combine the advantages of precision medicine with potent tumoricidal effects while mitigating the side effects of ADC and antibody-based RDC. The strong binding affinity of radioligand DOTA-AC-SP for Nectin-4 was verified using molecular docking and flow cytometry. The Nectin-4-targeted therapeutic [¹⁷⁷Lu]Lu- DOTA-AC-SP with high labeling efficiency(> 95%) and in vitro stability(retaining a radiochemical purity of > 90% after 70 h in PBS), was prepared via a one-step chelation reaction. Benefiting from the specific targeting property of DOTA-AC-SP, the [¹⁷⁷Lu]Lu- DOTA-AC-SP can bind to cells specifically with nanomolar affinity and can also rapidly accumulate in tumors with high Nectin-4 expression in vivo, consequently resulting in a favorable target-to-non-target ratio. Compared with the reported diagnostic peptide-based radiopharmaceuticals, such as [⁶⁸Ga]Ga-N188 [¹⁷⁷Lu]Lu- DOTA-AC-SP exhibits a similar high affinity (K_d = 2.08 nM vs. 2.45 nM of [⁶⁸Ga]Ga-N188). Since the design of [⁶⁸Ga]Ga-N188 takes into account the rapid clearance of the drug, no radioactive signal was observed at 1 h in the PET/CT imaging. In contrast, [¹⁷⁷Lu]Lu- DOTA-AC-SP can be retained in tumors for more than 36 h, which provides a basis for its therapeutic application.

The antitumor efficacy and survival impact of [¹⁷⁷Lu]Lu-DOTA-AC-SP were also evaluated in mice bearing SW780 tumors with high expression of Nectin-4. Although [¹⁷⁷Lu]Lu alone induced a certain degree of tumor necrosis, its therapeutic effect was limited due to non-targeted distribution. So, the significant mortality was also observed in the ¹⁷⁷Lu group, indicating potential safety concerns. In contrast, the group treated with [¹⁷⁷Lu]Lu-DOTA-AC-SP exhibited marked dose-dependent antitumor activity and improved survival outcomes.

Immunohistochemical analysis of resected tumor tissues revealed a significant reduction in Nectin-4 expression following [¹⁷⁷Lu]Lu-DOTA-AC-SP treatment, which is attributed to the binding of [¹⁷⁷Lu]Lu-DOTA-AC-SP to Nectin-4 on tumor cell surfaces, enabling β⁻ particles emitted from the decay of [¹⁷⁷Lu]Lu to induce direct DNA damage—including strand breaks, base lesions, and cross-links—leading to tumor cell death. Biodistribution studies conducted 14 days post-treatment showed significant retention of [¹⁷⁷Lu]Lu-DOTA-AC-SP within tumor tissues, further supporting its enhanced antitumor efficacy. Furthermore, preclinical safety evaluations, including HE Staining, hematological and biochemical parameter determination, body weight, behavior, and survival status observation, also demonstrated that [¹⁷⁷Lu]Lu-DOTA-AC-SP did not induce significant toxic effects on major normal organs (such as kidneys, liver, and bone marrow) at therapeutic doses. All these results demonstrate the efficacy and safety of [¹⁷⁷Lu]Lu-DOTA-AC-SP therapy.

Conclusion

[¹⁷⁷Lu]Lu-DOTA-AC-SP, a peptide-based therapeutic radiopharmaceuticals, targeting Nectin-4 was developed in this study. It not only exhibited favorable therapeutic efficacy but also demonstrated good safety profiles. To translate this potential into definitive clinical value, the focus of subsequent work should be on advancing systematic clinical translation research to ultimately confirm its efficacy and safety in patient populations.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

Nectin-4

Nectin cell adhesion molecule 4

UC

Urothelial carcinoma

EV

Enfortumab-vedotin

ADC

Antibody-drug Conjugates

RLTs

Radioligand therapies

RDC

Radionuclide-conjugated Drugs

SPECT/CT

Single-Photon Emission Computed Tomography/Computed Tomography

PSMA

Prostate Specific Membrane Antigen

SSTR

Somatostatin Receptors

PDB

Protein Data Bank

T/NT

Tumor/non-tumor ratios

T/M

Tumor/muscle ratios

T/K

Tumor/kidney ratios

PBS

Phosphate-buffered saline

FBS

Fetal bovine serum

radio-TLC

Radio-thin layer chromatography

Radio-HPLC

Radio-high performance liquid chromatography

H&E

Hematoxylin and eosin

IHC

Immunohistochemical

ALT

Alanine aminotransferase

BUN

Blood urea nitrogen

DAB

Diaminobenzidine

Author contributions

D.L., Y.Z., C.M., X.C. and X.L. made substantial contributions to the conceptualization of the work. Investigation was performed by M.C., methodology was performed by M.C. and X.L. The original draft of the manuscript was written by D.L. and Y.Z. Writing – review and editing was performed by D.L., W.C. and Z.L. All authors read and approved the final manuscript.

Funding

This work was supported by Strategic Priority Research Program of Chinese Academy of Sciences (XDA0580000), Joint Funds for the Innovation of Science and Technology, Fujian Province (2023Y9602), Natural Science Foundation Youth Project of Fujian Province (2023J0111191), and Key Project of Frontier Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences (SINAP-QYJS-202401).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The experimental animal protocol (Vista-IA-2-1-2306-01) was approved by the Animal Ethics Committee. This research did not involve human subjects, their data, or biological materials.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.