Skip to main content
NCBI home page
Springer logoLink to Springer
. 2025 May 20;52(12):4497–4510. doi: 10.1007/s00259-025-07238-7

Preclinical evaluation of [211At]At-AuNP-ABDMPL16 for targeted alpha therapy in Melanoma

Jiajia Zhang 1,2,#, Shanshan Qin 1,2,#, Xuhao Huang 3,4, Erina Hilmayanti 3, Fan Hu 1,2, Xiaohui Luan 1,2, Tianzhen Ye 7, Feize Li 7, Yuanyou Yang 7, Ning Liu 7, Kazuya Kabayama 3,5,6, Koichi Fukase 3,5,6, Fei Yu 1,2,✉
PMCID: PMC12491104  PMID: 40394402

Abstract

Purpose

The aim of this study is to overcome the challenges of poor tumor penetration and systemic toxicity in targeted alpha therapy (TAT) while also evaluating its immunomodulatory effects to enhance antitumor immune responses in melanoma treatment.

Methods

This study developed a 211At-labeled single-domain antibody agent ([211At]At-AuNP-ABDMPL16) targeting PD-L1, a protein overexpressed in melanoma cells. The binding affinity and internalization of [211At]At-AuNP-ABDMPL16 were evaluated in vitro using melanoma cell lines. In vivo studies in melanoma-bearing mice were conducted to assess biodistribution, pharmacokinetics, therapeutic efficacy, and the immune response induced by the treatment.

Results

[211At]At-AuNP-ABDMPL16 demonstrated high binding affinity and efficient internalization in melanoma cells, resulting in significant tumor cell death through α-particle radiation. In vivo, [211At]At-AuNP-ABDMPL16 preferentially accumulated in tumors, inhibited tumor growth, and prolonged survival in melanoma-bearing mice. The treatment also triggered a robust anti-tumor immune response, marked by increased cytotoxic T lymphocytes and reduced regulatory T cells within the tumor microenvironment, with minimal systemic toxicity.

Conclusion

[211At]At-AuNP-ABDMPL16 shows promise as a novel therapeutic for melanoma, combining effective tumor targeting with potent cytotoxic and immune-activating effects. These findings support further investigation of this 211At-labeled single-domain antibodies in clinical applications.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00259-025-07238-7.

Keywords: Targeted α-Therapy, 211At, Single-domain antibodies, Immune responses

Introduction

Melanoma, known for its high metastatic potential, continues to rise in global incidence. While early surgical resection can significantly improve prognosis, advanced-stage patients often face the failure of traditional chemotherapy and radiotherapy [1–3]. In this context, there is an urgent need to develop new therapeutic strategies to improve overall survival and quality of life for patients. Recent studies have found that, beyond directly killing tumors, radiation therapy can also induce immunogenic cell death (ICD), releasing damage-associated molecular patterns (such as calreticulin and HMGB1), which activate dendritic cells and cytotoxic T lymphocytes, thereby triggering antitumor immunity [4, 5]. The direct killing—immune activation dual mechanism makes radiation therapy a core component of combination immunotherapy strategies [6–8].

Targeted alpha therapy (TAT) has unique advantages due to the high linear energy transfer (LET) of alpha particles (80–100 keV/μm), which efficiently induce DNA double-strand breaks, while the short range (50–100 μm) minimizes damage to normal tissues [9–11]. Among various alpha-emitting radionuclides, 211At stands out due to its 7.2-h half-life and stable chemical binding properties, overcoming the limitations of 225Ac (redistribution of daughter radionuclides) and 223Ra (bone tissue accumulation) [12–14]. However, the efficacy of TAT heavily depends on precise targeting vehicles [15–17]. Monoclonal antibodies have poor tumor penetration due to their large molecular weight and may cause blood toxicity, while peptides are rapidly cleared, limiting their sustained therapeutic effects [18–20]. Single-domain antibodies (sdAbs), with their small size (15 kDa) and nanomolar affinity, show unique advantages in tumor-targeted therapy [21–24]. Incorporating albumin-binding domain (ABD) into sdAbs extends their circulation time, increases tumor uptake, and enhances imaging contrast and therapeutic potential by binding to endogenous albumin [25, 26].

PD-L1 is overexpressed in the melanoma, which provides it with dual therapeutic potential: it not only directly targets tumor cells but also blocks the PD- 1/PD-L1 immune checkpoint signal [27–29]. Although 131I-labeled PD-L1 sdAbs have shown preclinical efficacy [30], alpha-emitting radionuclides like 211At may enhance immunogenicity through radiation by releasing tumor antigens and preserving lymphocyte infiltration, thereby amplifying therapeutic effects [31–34]. However, the exact mechanisms by which TAT reshapes the tumor microenvironment remain to be elucidated.

Hence, this study constructed an ABD-modified single-domain antibody targeting PD-L1 (ABDMPL16) and labeled it with 211At, using gold nanoparticles (AuNP) as a chelator for TAT. The primary objective was to evaluate the therapeutic efficacy of [211At]At-AuNP-ABDMPL16 in melanoma and its potential in activating immune responses (Scheme 1). To assess its safety and effectiveness, both in vivo and in vitro experiments were conducted, while the underlying mechanisms promoting antitumor immune responses were also explored. This research not only demonstrates the potential of [211At]At-AuNP-ABDMPL16 in melanoma therapy but also paves the way for precision cancer treatment by enhancing both direct cytotoxicity and immune-mediated antitumor effects, thus providing a theoretical foundation for its clinical translation.

Scheme 1.

Scheme 1

Open in a new tab

The schematic diagram of the preparation of [211At]At-AuNP-ABDMPL16 and its role in triggering systemic immune responses (By Figdraw). CTL: Cytotoxic T Lymphocytes; Mature DC: Mature Dendritic Cells; Immature DC: Immature Dendritic Cells; TAAs: Tumor-Associated Antigens

Results

Binding and internalization abilities of ABDMPL16

A novel mouse PD-L1 specific single-domain antibodies (ABDMPL16) was expressed. Results showed that ABDMPL16 has a molecular weight of about 21 kDa (Fig. 1a). The binding kinetics of ABDMPL16 to PD-L1 were measured using surface plasmon resonance (SPR). A series of increasing concentrations of ABDMPL16 (from 0.78125 nM to 50 nM) demonstrated a concentration-dependent increase in binding response. The calculated dissociation constant (Kd) of 2.582 nM indicates a high binding affinity of ABDMPL16 for PD-L1, suggesting that it can specifically target PD-L1-expressing cells with strong interaction (Fig. 1b). Flow cytometry was employed to evaluate the binding of AF488-labeled ABDMPL16 to PD-L1 on cells. At both high and low concentrations of AF488-ABDMPL16, there was a significant shift in fluorescence intensity compared to the control group, further confirming that ABDMPL16 binds effectively to PD-L1-expressing cells in a concentration-dependent manner (Fig. 1c). The binding capability of ABDMPL16 was further demonstrated by immunofluorescence staining (Fig. 1d). Cells treated with AF488-ABDMPL16 exhibited strong PD-L1 signal, as observed in the merged images. Quantification of the positive area percentage showed a significant increase in PD-L1 staining in cells treated with AF488-ABDMPL16 compared to the control (p < 0.01), confirming the specific targeting of PD-L1 by ABDMPL16.

Fig. 1.

Fig. 1

Open in a new tab

Binding and Internalization Abilities of ABDMPL16. (a) Purity of ABDMPL16 analyzed by sulfate polyacrylamide-gel electrophoresis (SDS-PAGE). (b) Surface plasmon resonance (SPR) binding curves of ABDMPL16 to PD-L1 at varying concentrations. (c) Flow cytometry analysis of PD-L1 binding by AF488-ABDMPL16 at low and high concentrations. (d) Represent image and quantification of binding ability of ABDMPL16 (n = 3). (e) Transmission electron microscopy (TEM) images of AuNP-ABDMPL16. (f) Internalization ability of AuNP-ABDMPL16 by goldenhancement assay. Statistical significance between groups was calculated using Students t-test, with **p < 0.01

To efficiently, mildly, and conveniently conjugate 211At and ABDMPL16, NHS-activated gold nanoparticles (AuNP) were employed. Transmission electron microscopy (TEM) was used to characterize the AuNP, revealing a particle diameter of approximately 5 nm (Fig. S1). Subsequently, dynamic light scattering (DLS) measurement showed a hydrated particle size of 20.37 nm (Fig. S2), which is larger than the TEM-measured diameter due to the formation of a solvation layer around the particles caused by solvent molecule adsorption. TEM imaging of the AuNP-ABDMPL16 nanoparticles (Fig. 1e) revealed uniformly distributed particles with an average diameter of approximately 10 nm, while DLS measurements indicated a hydrated particle size of 30.22 nm (Fig. S3), confirming the successful conjugation of ABDMPL16 to the AuNP. MALDI-TOF MS analysis further validated the conjugation, as indicated by a significant shift in peak values, confirming the successful attachment of ABDMPL16 to AuNP (Fig. S4).

Additionally, the cellular internalization ability of AuNP-ABDMPL16 was assessed using a gold enhancement assay (Fig. 1f, Fig. S5). The results showed that in B16 F10 cells, the accumulation of nanoparticles was highest in the high concentration group. However, when PD-L1 was blocked by ABDMPL16, the uptake of AuNP-ABDMPL16 was significantly reduced. This finding demonstrates that the internalization of the nanoparticles was specifically mediated through PD-L1 binding.

In vitro targeted α-therapy using [211At]At-AuNP-ABDMPL16

The synthesis of [211At]At-AuNP-ABDMPL16 is illustrated in Fig. 2a. The successful synthesis of [211At]At-AuNP-ABDMPL16 was confirmed, with the radiolabeling yield exceeding 90% (Fig. 2b). The radiolabeling stability of [211At]At-AuNP-ABDMPL16 was maintained over time in FBS or PBS, demonstrating robustness even after nearly three half-lives in physiological conditions (Fig. 2c). In vitro cytotoxicity assays revealed that 211At reduced cell viability (53.82 ± 4.29% at 0.5 Mbq/mL) (Fig. 2d), [211At]At-AuNP-ABDMPL16 treatment led to a significant dose-dependent reduction in B16 F10 cell viability, with viability decreasing to 29.98 ± 7.19% at a dose of 0.5 Mbq/mL (Fig. 2e). The effect of free 211At was less pronounced than that of [211At]At-AuNP-ABDMPL16, underscoring the enhanced efficacy of targeted alpha therapy in further reducing tumor activity. Time-dependent cellular uptake of [211At]At-AuNP-ABDMPL16. The uptake of [211At]At-AuNP-ABDMPL16 was significantly higher at 1 h compared to free 211At. Blocking studies showed a reduction in uptake (Fig. 2f), confirming receptor-mediated endocytosis.

Fig. 2.

Fig. 2

Open in a new tab

In Vitro Targeted α-Therapy Using [211At]At-AuNP-ABDMPL16. (a) Preparation of [211At]At-AuNP-ABDMPL16. (b) Labeling yield of [211At]At-AuNP-ABDMPL16. (c) Radiolabeling stability of [211At]At-AuNP-ABDMPL16 co-incubated in FBS or PBS over time. (d, e) Relative viability of B16 F10 cells treated with 211At and [211At]At-AuNP-ABDMPL16 at different doses for 24 h. (f) Cell binding assay for determination of the 211At, and [211At]At-AuNP-ABDMPL16, and after blocking with ABDMPL16. (g) Representative immunofluorescence micrographs of γ-H2 AX expression in B16 F10 cells after different treatments. (h) Representative immunofluorescence micrographs of Fas expression in B16 F10 cells after different treatments. Data are presented as mean ± SD (n = 3). G1-G4 represent Control, AuNP-ABDMPL16, 211At, and [211At]At-AuNP-ABDMPL16

As a sensitive radiation biomarker, γ-H2 AX is highly reliable for detecting DNA damage and repair mechanisms. B16 F10 cells were divided into five groups receiving varying doses of [211At]At-AuNP-ABDMPL16 (0, 0.01, 0.1, 0.5, 1 Mbq/mL). Immunofluorescence staining demonstrated increased γ-H2 AX expression following [211At]At-AuNP-ABDMPL16 treatment, indicating elevated DNA damage (Fig. 2g). Upon recognition of target cells, cytotoxic T lymphocytes (CTLs) initiate apoptosis in tumor cells through the interaction between Fas ligand and Fas receptors on the tumor cell surface. The observed increase in Fas expression after 211At and [211At]At-AuNP-ABDMPL16 irradiation, contrasted with the unchanged Fas levels in the AuNP-ABDMPL16-only group (Fig. 2h), aligns with findings from other studies, reinforcing the effectiveness of targeted alpha therapy in inducing tumor cell apoptosis.

Pharmacokinetics and biodistribution of [211At]At-AuNP-ABDMPL16

The pharmacokinetic analysis of [211At]At-AuNP-ABDMPL16 was performed by monitoring the radioactivity concentration in blood over time (Fig. 3a). The curve indicates a biphasic clearance pattern, with rapid clearance in the initial phase followed by a slower elimination phase. The pharmacokinetic analysis of the radiotracer demonstrated distinct distribution and elimination phases, as shown in Fig. 3b. The calculated alpha half-life (Alpha_HL) was 6.34 ± 3.24 min, reflecting the rapid distribution phase, while the beta half-life (Beta_HL) was 694 ± 108 min, indicating a slower elimination phase. The total area under the curve (AUC) was determined to be (3.76 ± 0.43) × 109 Bq·min/L, suggesting substantial exposure to the radiotracer over time. Additionally, the area under the first moment curve (AUMC) was (3.69 ± 0.99) × 1012 Bq·min2/L, which, combined with the mean residence time (MRT) of 982 ± 154 min, highlights the extended retention of the radiotracer. The maximum concentration (C_max) reached (1.17 ± 0.43) × 107 Bq/L, demonstrating a high peak plasma level following administration. Clearance (CL) was estimated at (1.86 ± 0.21) × 10–5 L/min, consistent with a prolonged elimination profile. These findings collectively provide detailed insights into the biodistribution, systemic exposure, and clearance of the radiotracer.

Fig. 3.

Fig. 3

Open in a new tab

Pharmacokinetics and Biodistribution of [211At]At-AuNP-ABDMPL16. (a, b) Pharmacokinetics of [211At]At-AuNP-ABDMPL16. (c-e) Biodistribution of [211At]At-AuNP-ABDMPL16 in healthy C57BL/6 mice at different time points (3 h, 7 h, 24 h). Data are presented as mean ± SD (n = 4). (f, g) Biodistribution of [211At]At-AuNP-ABDMPL16 in melanoma-bearing C57BL/6 mice at different time points (7 h, 24 h). Data are presented as mean ± SD (n = 3)

The biodistribution of [211At]At-AuNP-ABDMPL16 was evaluated in normal mice at 3, 7, and 24 h post-injection (Fig. 3c, d, and e). At 3 h, moderate uptake was observed in the liver, spleen, and lungs, which is consistent with the biological filtration and metabolic functions of these organs. By 7 h, liver uptake increased significantly, indicating clearance through the liver, while uptake in the spleen and lungs remained relatively stable. At 24 h, the radiotracer was still retained in the liver and spleen, suggesting that these organs play a key role in the clearance of [211At]At-AuNP-ABDMPL16.The uptake of [211At]At-AuNP-ABDMPL16 in the stomach is observed to a certain extent, suggesting that the nonspecific uptake of the radiotracer in the stomach may be related to the high blood flow to the gastric mucosa. The thyroid, due to the high expression of sodium/iodide symporter (NIS), has an affinity for certain radiolabeled iodine isotopes, such as 211At. In this study, the uptake of [211At]At-AuNP-ABDMPL16 in the thyroid was observed at certain levels at 3, 7, and 24 h, with no significant changes.

In the tumor-bearing model, the biodistribution analysis at 7 and 24 h (Figs. 3f and g) showed substantial accumulation of [211At]At-AuNP-ABDMPL16 in the tumor, with higher uptake at 7 h (20.64 ± 4.71%ID/g), significantly higher than in most normal tissues. At 24 h, tumor uptake slightly decreased. These results suggest that [211At]At-AuNP-ABDMPL16 achieves effective tumor targeting and retention, while also demonstrating a pharmacokinetic profile indicative of its clearance predominantly through the liver and spleen. The differential uptake between tumor and normal tissues highlights the potential for this radiotracer in targeted alpha therapy.

In vivo antitumor efficacy of targeted α-therapy in B16 F10 tumor models

Based on the favorable results from the in vitro and biodistribution experiments, we further evaluated tumor volume, body weight changes, HE staining, and apoptosis in tumor tissues across different treatment groups to verify the antitumor efficacy. Tumor volume changes over time among the different treatment groups (Fig. 4a). Tumor volumes in the control and AuNP-ABDMPL16 groups increased significantly within a short period, while tumor growth in the [211At]At-AuNP-ABDMPL16 group was delayed, indicating that this treatment was more effective at inhibiting tumor growth compared to 211At alone. There were also differences in body weight changes among the groups (Fig. 4b). The body weight of mice in the control group increased slightly as the tumors grew, whereas the body weight in the 211At and [211At]At-AuNP-ABDMPL16 groups remained relatively stable, with the [211At]At-AuNP-ABDMPL16 group exhibiting the best tolerance. The survival curves for each group were showed in Fig. 4c. Mice in the [211At]At-AuNP-ABDMPL16 group exhibited significantly prolonged survival compared to the 211At and ABDMPL16 groups. The median survival times were 8 days for both the control and ABDMPL16 groups, 12 days for the 211At group, and notably, 24 days for the [211At]At-AuNP-ABDMPL16 group, demonstrating a significant improvement. On day 10 of treatment, the tumor volumes in the [211At]At-AuNP-ABDMPL16 group exhibited slow growth, whereas tumors in the 211At group continued to enlarge, further demonstrating the effectiveness of the combined treatment (Fig. 4d).

Fig. 4.

Fig. 4

Open in a new tab

In Vivo Antitumor Evaluations of Targeted α-Therapy in B16 F10 Tumor Models. (a) Average tumor growth curves of B16 F10 tumor-bearing mice from different treatment groups (n = 5). (b) Time-dependent body weight changes in mice after different treatments (n = 5). (c) Kaplan–Meier survival curves of mice receiving various therapies. (d) Representative images of tumors. (e, f) Representative H&E histopathology and TUNEL staining of tumors. (g) Representative H&E-stained tissue sections from animals in each group

HE staining revealed dense tumor cells with significant mitotic structures in the control group. In contrast, the [211At]At-AuNP-ABDMPL16 group showed a marked reduction in tumor cells and an expansion of necrotic areas, indicating effective tumor cell necrosis induced by this treatment (Fig. 4e). TUNEL staining results showed a significant increase in tumor cell apoptosis in the [211At]At-AuNP-ABDMPL16 group, with stronger apoptotic signals than those observed in the 211At or AuNP-ABDMPL16 groups alone (Fig. 4f), further confirming the apoptosis-inducing effect of the combined treatment. Finally, HE staining of major organs (heart, liver, spleen, lung, kidney) in all treatment groups revealed no significant organ damage (Fig. 4g), indicating the safety and good biocompatibility of the treatments.

Immune responses induced by targeted α-therapy

To further explore the immune response changes in the tumor microenvironment (TME) following [211At]At-AuNP-ABDMPL16 treatment, we assessed variations in different immune cell populations using flow cytometry. The result illustrates the differences among the control group, the 211At alone treatment group, and the [211At]At-AuNP-ABDMPL16 treatment group in CD45+CD3+CD8+ T cells, regulatory T cells (Tregs), and dendritic cells (DCs) expressing the co-stimulatory molecules CD80 and CD86. As shown in Fig. 5a, compared to the control group, the proportion of CD3+CD8+ T cells significantly increased in the [211At]At-AuNP-ABDMPL16 treatment group. Statistical analysis indicated that after [211At]At-AuNP-ABDMPL16 treatment, this proportion rose from 29.95 ± 4.34% to 46.69 ± 1.19% (p < 0.01) (Fig. 5b). This finding suggests that the treatment might promote T cell activation or recruitment. In the analysis of regulatory T cells, the [211At]At-AuNP-ABDMPL16 treatment group showed a significant decrease in the proportion of Foxp3+ cells, from 3.16 ± 1.43% in the control group to 0.7 ± 0.18% (p < 0.05) (Fig. 5c, d). This might reflect a weakening of the immunosuppressive environment in the TME, potentially enhancing the anti-tumor immune response.

Fig. 5.

Fig. 5

Open in a new tab

In Vivo Anti-Tumor Immune Responses. (a, b) Representative flow cytometry plots showing CTL cells (CD45+CD3+CD8+) in B16 F10 tumors and their relative quantification (n = 3). (c, d) Representative flow cytometry plots showing regulatory T cells (CD45+CD3+CD4+Foxp3+) in B16 F10 tumors and their relative quantification (n = 3). (e–h) Representative flow cytometry plots showing DC cells (CD45+CD11c+CD80+, CD45+CD11c+CD86+) in B16 F10 tumors and their relative quantification (n = 3). Statistical significance between groups was calculated using Students t-test, with *p < 0.05, **p < 0.01

Furthermore, we analyzed the expression of the co-stimulatory molecules CD80 and CD86 on dendritic cells (Fig. 5e and g). The results revealed a significant increase in the proportion of CD11c+CD80+ DCs in the [211At]At-AuNP-ABDMPL16 treatment group, reaching 19.97 ± 2.33% (p < 0.05) (Fig. 5f), while the proportion of CD11c+CD86+ DCs rose to 18.05 ± 1.69% (p < 0.01) (Fig. 5h). These data suggest that this treatment may enhance the antigen-presenting capacity of dendritic cells, thereby promoting T cell-mediated immune responses. In conclusion, these results demonstrate the regulatory effects of [211At]At-AuNP-ABDMPL16 on immune cells within the tumor microenvironment, highlighting its potential as a promising strategy for radio-immunotherapy.

Long-term safety of [211At]At-AuNP-ABDMPL16

We evaluated the long-term toxicity of [211At]At-AuNP-ABDMPL16 in normal C57BL/6 mice. Figure 6a shows the trend in body weight changes during the experiment. From baseline to the end of the experiment, the body weight of mice gradually increased, with no significant difference observed between the control group and the [211At]At-AuNP-ABDMPL16 group, indicating that the treatment did not significantly affect the normal growth and development of the mice. Next, we analyzed changes in hematological parameters to further assess the safety of the [211At]At-AuNP-ABDMPL16.

Fig. 6.

Fig. 6

Open in a new tab

Long-Term In Vivo Safety of Targeted α-Therapy. (a) Time-dependent body weight changes in healthy mice after different treatments (n = 5). (b-k) Blood routine parameters, including white blood cell (WBC), red blood cell (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), platelet (PLT), mean platelet volume (MPV), hemoglobin (HGB), and plateletcrit (PCT) were measured

Various blood parameters were compared between the control and treatment groups (Fig. 6b- 6k). The results show that white blood cell count (WBC), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), platelet count (PLT), mean platelet volume (MPV), hemoglobin concentration (HGB), and plateletcrit (PCT) did not show significant differences between the two groups. These results indicate that [211At]At-AuNP-ABDMPL16 did not have a significant adverse effect on the hematological parameters of mice during the treatment, further supporting the biosafety of this single-domain antibodies.

Discussion

This study successfully developed and evaluated [211At]At-AuNP-ABDMPL16, demonstrating its significant potential in TAT for melanoma. By leveraging the high specificity and rapid tumor penetration of sdAbs, [211At]At-AuNP-ABDMPL16 overcomes the major limitations of existing radiopharmaceuticals, in terms of radiochemical stability, pharmacokinetics, and therapeutic efficiency, showcasing remarkable advantages [30, 35].

Efficient and stable conjugation conditions are required for successful labeling. Gold nanoparticle (AuNP), due to their easily modifiable surface, have been widely used as drug carriers for diagnostic and therapeutic agents. 211At can be combined with AuNP through direct adsorption or covalent bonding to form a stable radioactive nanocomplex. Previous studies have shown that 211At can achieve a high radiochemical yield (RCY) within 5 min when reacted with AuNP, without the need for a purification process. This reaction method, based on AuNPs, is rapid, efficient, and mild, not only optimizing the half-life utilization of 211At but also enabling precise targeted therapy through the enhanced permeability and retention effect [36].

Radiochemical stability is a critical factor in ensuring therapeutic efficacy. The radiochemical purity of [225Ac]Ac-HEHA-PD-L1-i rapidly declined to 64% within 72 h after labeling, primarily due to the recoil energy from 225Ac decay disrupting the bond between the radionuclide and the chelator [35]. In contrast, [211At]At-AuNP-ABDMPL16 maintained high stability over a similar period, attributed to the shorter half-life of 211At and the protective effect of the gold nanoparticles. This stability reduces radionuclide leakage, ensuring consistent therapeutic performance during treatment and addressing the instability issues observed in 225Ac-based therapies. Previous studies have also demonstrated that using gold nanostars (GNS) for radiolabeling with 211At exhibits excellent stability in vivo [37]. These studies further highlighted that 211At-labeled GNS showed minimal in vivo deastatination while achieving promising therapeutic efficacy, providing strong support for our findings.

Pharmacokinetic and biodistribution studies further validated the potential of [211At]At-AuNP-ABDMPL16 in precision therapy. The drug exhibited a biphasic clearance pattern, characterized by significant tumor uptake and prolonged tumor retention following the initial rapid clearance phase. This selective accumulation reduced off-target effects and significantly improved the therapeutic index. Biodistribution studies also revealed some accumulation of [211At]At-AuNP-ABDMPL16 in the liver and spleen, reflecting the natural clearance mechanism of the reticuloendothelial system. Additionally, the high radioactivity observed in the thyroid, consistent with the chemical similarity between 211At and iodine, was anticipated. The residual radioactivity in the stomach is likely due to the high blood flow in the gastric mucosa, which facilitates the absorption of substances, including 211At, rather than the instability of [211At]At-AuNP-ABDMPL16. Additionally, factors such as gastric motility and acid secretion may contribute to the accumulation of 211At in the stomach.

Building on these pharmacokinetic advantages, [211At]At-AuNP-ABDMPL16 demonstrated unique benefits in tumor targeting and delivery efficiency. In contrast, [225Ac]Ac-HEHA-PD-L1-i suffered from non-specific accumulation in the liver and spleen due to redistribution of its decay progeny, while [131I]I-Nb109 lacked a multifunctional carrier design, resulting in limited tumor retention and insufficient radiation dose delivery. By incorporating gold nanoparticles, [211At]At-AuNP-ABDMPL16 not only facilitated receptor-mediated endocytosis but also enhanced radiation dose delivery to the tumor, further prolonging tumor retention and reducing off-target radiation exposure, thereby consolidating its advantages in precision therapy.

In terms of therapeutic efficiency, the radiation properties of 211At were significantly superior to those of 131I and 225Ac. While [131I]I-Nb109 emitted β-particles with long penetration depth, its relatively low energy limited its ability to effectively kill micrometastases or isolated cancer cells. Furthermore, the mismatch between the physical half-life of 131I (8.04 days) and the biological half-life of Nb109 (1–2 h) resulted in premature radionuclide clearance before effective tumor accumulation, substantially reducing therapeutic efficiency [30]. In contrast, [211At]At-AuNP-ABDMPL16 emitted high-energy α-particles with a short range, enabling localized and highly effective killing of tumor cells by inducing DNA double-strand breaks while minimizing damage to surrounding healthy tissues [38, 39] The 7.2-h half-life of 211At further ensured optimal alignment with the pharmacokinetic profile of single-domain antibodies, significantly enhancing therapeutic outcomes.

In addition to directly killing tumor cells, TAT also induces immune responses. Previous studies have shown that alpha-emitting radionuclides such as 225Ac and 227Th can induce immunogenic cell death (ICD), leading to the release of tumor antigens and the activation of dendritic cells (DCs) and T cells [40, 41]. For example, 225Ac treatment in mouse models resulted in increased DC and CD8+ T cell activation, along with higher levels of ICD markers such as HMGB1 and ATP, and improved tumor infiltration of CD8+ T cells. Similarly, 227Th treatment increased antigen-presenting cells, especially mature DCs, and elevated PD-L1 expression in the tumor microenvironment. Consistent with these studies, our [211At]At-AuNP-ABDMPL16 treatment also showed significant immune-modulatory effects, particularly an increase in the proportion of CD8+ T cells and a decrease in Treg cells within the TME. Notably, [211At]At-AuNP-ABDMPL16 significantly enhanced the expression of co-stimulatory molecules CD80 and CD86 on DCs. This suggests that 211At not only kills tumor cells through the direct cytotoxicity of alpha particles but also reprograms the immunosuppressive tumor microenvironment to further enhance anti-tumor immune responses.

The long-term safety of [211At]At-AuNP-ABDMPL16, as evidenced by stable body weight and hematological parameters in treated mice, indicates minimal systemic toxicity, making it a promising candidate for further clinical development. This safety profile is particularly important for the clinical translation of alpha-emitting radiopharmaceuticals, as insufficient targeting can lead to significant side effects [42, 43].

However, this study has some limitations. Future studies should include additional control groups, such as [211At]At-AuNP or other non-PD-L1-targeted 211At radiopharmaceuticals, to more comprehensively assess the differences in targeting and therapeutic efficacy. More in vivo distribution time points should be added to better evaluate the distribution of [211At]At-AuNP-ABDMPL16 in the body. Additionally, the long-term effects of [211At]At-AuNP-ABDMPL16 on immune memory and the potential for immune evasion by tumors require further investigation. More detailed studies, such as single-cell RNA sequencing or multiplexed immunohistochemistry, are needed to elucidate the mechanisms underlying the enhanced immune activation observed with [211At]At-AuNP-ABDMPL16 treatment. Although our study focused on melanoma, future studies should explore the applicability of [211At]At-AuNP-ABDMPL16 in other tumor types with high PD-L1 expression.

Conclusion

This study highlights the potential of targeted α-therapy using [211At]-labeled AuNP-ABDMPL16 as a promising treatment for melanoma. The therapy not only demonstrated significant tumoricidal effects in vitro by enhancing DNA damage and promoting apoptosis in melanoma cells, but also showed a favorable safety profile in vivo. Additionally, the induction of anti-tumor immune responses suggests that this therapeutic approach could be further potentiated when combined with immunotherapy. These findings underscore the therapeutic value of [211At]At-AuNP-ABDMPL16 and warrant further investigation in preclinical and clinical settings to fully realize its potential as an effective treatment for melanoma.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOC 23685 kb) (23.1MB, doc)
Supplementary file2 (PNG 161 kb) (161.7KB, png)

Author contribution

Conception and design: Jiajia Zhang, Shanshan Qin. Development of methodology: Jiajia Zhang, Shanshan Qin, Xuhao Huang, Erina Hilmayanti. Acquisition of data: Jiajia Zhang, Shanshan Qin, Fan Hu, Xiaohui Luan, Tianzhen Ye. Analysis and interpretation of data: Jiajia Zhang. Writing, review, and/or revision of the manuscript: Jiajia Zhang, Shanshan Qin, Feize Li, Yuanyou Yang, Ning Liu. Study supervision: Kazuya Kabayama, Koichi Fukase and Fei Yu.

Funding

This work was supported by National Natural Science Foundation of China (Grant No. 82272030), the construction project of Shanghai Key Laboratory of Molecular Imaging (Grant No.18DZ2260400), Program of China Scholarship Council (Grant No. 202306260219).

Data availability

Original data are available on request.

Declarations

Conflicts of interest

The authors have no conflict of interest or competing interest to declare.

Ethics approval

All animal experiments were approved by Animal Welfare Ethics Committee of Shanghai Tenth People’s Hospital with an approval number (ID: SHDSYY- 2024–0746).

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jiajia Zhang and Shanshan Qin contributed equally to this work.

References

  • 1.Bhave P, Hong A, Lo SN, et al. Efficacy and toxicity of adjuvant radiotherapy in recurrent melanoma after adjuvant immunotherapy. J Immunother Cancer. 2023;11(3):e006629. 10.1136/jitc-2022-006629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Gorry C, McCullagh L, O’Donnell H, et al. Neoadjuvant treatment for stage III and IV cutaneous melanoma. Cochrane Database Syst Rev. 2023;1(1):Cd012974. 10.1002/14651858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Reijers ILM, Menzies AM, van Akkooi ACJ, et al. Personalized response-directed surgery and adjuvant therapy after neoadjuvant ipilimumab and nivolumab in high-risk stage III melanoma: the PRADO trial. Nat Med. 2022;28(6):1178–88. 10.1038/s41591-022-01851-x. [DOI] [PubMed] [Google Scholar]
  • 4.Jagodinsky JC, Vera JM, Jin WJ, et al. Intratumoral radiation dose heterogeneity augments antitumor immunity in mice and primes responses to checkpoint blockade. Sci Transl Med. 2024;16(765):eadk0642. 10.1126/scitranslmed.adk0642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Zhang Q, Jiang L, Wang W, et al. Potentiating the radiation-induced type I interferon antitumoral immune response by ATM inhibition in pancreatic cancer. JCI Insight. 2024;9(6):e168824. 10.1172/jci.insight.168824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Bao X, Xie L. Targeting purinergic pathway to enhance radiotherapy-induced immunogeniccancer cell death. J Exp Clin Cancer Res. 2022;41(1):222. 10.1186/s13046-022-02430-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Sun Q, Li J, Ding Z, Liu Z. Radiopharmaceuticals heat anti-tumor immunity. Theranostics. 2023;13(2):767–86. 10.7150/thno.79806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Luo R, Onyshchenko K, Wang L, et al. Necroptosis-dependent immunogenicity of Cisplatin: Implications for enhancing the radiation-induced Abscopal effect. Clin Cancer Res. 2023;29(3):667–83. 10.1158/1078-0432.Ccr-22-1591. [DOI] [PubMed] [Google Scholar]
  • 9.Ye T, Yu Y, Qu G, et al. (211)At radiolabeled APBA-FAPI for enhanced targeted-alpha therapy of glioma. Eur J Med Chem. 2024;279:116919. 10.1016/j.ejmech.2024.116919. [DOI] [PubMed] [Google Scholar]
  • 10.Bidkar AP, Wang S, Bobba KN, et al. Treatment of prostate cancer with CD46-targeted 225Ac alpha particle radioimmunotherapy. Clin Cancer Res. 2023;29(10):1916–28. 10.1158/1078-0432.Ccr-22-3291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Chung SK, Vargas DB, Chandler CS, et al. Efficacy of HER2-targeted intraperitoneal (225)Ac α-pretargeted radioimmunotherapy for small-volume ovarian peritoneal carcinomatosis. J Nucl Med. 2023;64(9):1439–45. 10.2967/jnumed.122.265095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Kaneda-Nakashima K, Zhang Z, Manabe Y, et al. α-Emitting cancer therapy using (211) At-AAMT targeting LAT1. Cancer Sci. 2021;112(3):1132–40. 10.1111/cas.14761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Echigo H, Mishiro K, Munekane M, et al. Development of probes for radiotheranostics with albumin binding moiety to increase the therapeutic effects of astatine-211 ((211)At). Eur J Nucl Med Mol Imaging. 2024;51(2):412–21. 10.1007/s00259-023-06457-0. [DOI] [PubMed] [Google Scholar]
  • 14.Roncali L, Marionneau-Lambot S, Roy C, et al. Brain intratumoural astatine-211 radiotherapy targeting syndecan-1 leads to durable glioblastoma remission and immune memory in female mice. EBioMedicine. 2024;105:105202. 10.1016/j.ebiom.2024.105202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Chakravarty R, Song W, Chakraborty S, Cai W. Fibroblast activation protein (FAP)-targeted radionuclide therapy: which ligand is the best? Eur J Nucl Med Mol Imaging. 2023;50(10):2935–9. 10.1007/s00259-023-06338-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Ruigrok EAM, van Vliet N, Dalm SU, et al. Extensive preclinical evaluation of lutetium-177-labeled PSMA-specific tracers for prostate cancer radionuclide therapy. Eur J Nucl Med Mol Imaging. 2021;48(5):1339–50. 10.1007/s00259-020-05057-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Yang T, Peng L, Qiu J, et al. A radiohybrid theranostics ligand labeled with fluorine-18 and lutetium-177 for fibroblast activation protein-targeted imaging and radionuclide therapy. Eur J Nucl Med Mol Imaging. 2023;50(8):2331–41. 10.1007/s00259-023-06169-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Korsen JA, Gutierrez JA, Tully KM, et al. Delta-like ligand 3-targeted radioimmunotherapy for neuroendocrine prostate cancer. Proc Natl Acad Sci U S A. 2022;119(27):e2203820119. 10.1073/pnas.2203820119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Zboralski D, Hoehne A, Bredenbeck A, et al. Preclinical evaluation of FAP-2286 for fibroblast activation protein targeted radionuclide imaging and therapy. Eur J Nucl Med Mol Imaging. 2022;49(11):3651–67. 10.1007/s00259-022-05842-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.King AP, Gutsche NT, Raju N, et al. (225)Ac-MACROPATATE: A novel α-particle peptide receptor radionuclide therapy for neuroendocrine tumors. J Nucl Med. 2023;64(4):549–54. 10.2967/jnumed.122.264707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Feng Y, Meshaw R, Zhao XG, et al. Effective treatment of human breast carcinoma xenografts with single-dose (211)At-Labeled Anti-HER2 single-domain antibody fragment. J Nucl Med. 2023;64(1):124–30. 10.2967/jnumed.122.264071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Feng Y, Sarrett SM, Meshaw RL, et al. Site-specific radiohalogenation of a HER2-targeted single-domain antibody fragment using a novel residualizing prosthetic agent. J Med Chem. 2022;65(22):15358–73. 10.1021/acs.jmedchem.2c01331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Yu X, Long Y, Chen B, et al. PD-L1/TLR7 dual-targeting nanobody-drug conjugate mediates potent tumor regression via elevating tumor immunogenicity in a host-expressed PD-L1 bias-dependent way. J Immunother Cancer 2022; 10:10e004590. 10.1136/jitc-2022-004590. [DOI] [PMC free article] [PubMed]
  • 24.Hu B, Liu T, Li L, et al. IgG-binding nanobody capable of prolonging nanobody-based radiotracer plasma half-life and enhancing the efficacy of tumor-targeted radionuclide therapy. Bioconjug Chem. 2022;33(7):1328–39. 10.1021/acs.bioconjchem.2c00209. [DOI] [PubMed] [Google Scholar]
  • 25.Zhang Y, Zhang D, An S, et al. Development and characterization of nanobody-derived CD47 theranostic pairs in solid tumors. Research (Wash D C) 2023;6:0077. 10.34133/research.0077. [DOI] [PMC free article] [PubMed]
  • 26.An S, Zhang D, Zhang Y, et al. GPC3-targeted immunoPET imaging of hepatocellular carcinomas. Eur J Nucl Med Mol Imaging. 2022;49(8):2682–92. 10.1007/s00259-022-05723-x. [DOI] [PubMed] [Google Scholar]
  • 27.VanderWalde A, Bellasea SL, Kendra KL, et al. Ipilimumab with or without nivolumab in PD-1 or PD-L1 blockade refractory metastatic melanoma: a randomized phase 2 trial. Nat Med. 2023;29(9):2278–85. 10.1038/s41591-023-02498-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Serratì S, Guida M, Di Fonte R, et al. Circulating extracellular vesicles expressing PD1 and PD-L1 predict response and mediate resistance to checkpoint inhibitors immunotherapy in metastatic melanoma. Mol Cancer. 2022;21(1):20. 10.1186/s12943-021-01490-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Zhang Y, Cao M, Wu Y, et al. Preclinical development of novel PD-L1 tracers and first-in-human study of [(68)Ga]Ga-NOTA-RW102 in patients with lung cancers. J Immunother Cancer. 2024;12(4):e008794. 10.1136/jitc-2024-008794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Zhu M, Zhang J, Yang M, et al. In vitro and in vivo study on the treatment of non-small cell lung cancer with radionuclide labeled PD-L1 nanobody. J Cancer Res Clin Oncol. 2023;149(11):8429–42. 10.1007/s00432-023-04793-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Link EM, Carpenter RN. 211At-methylene blue for targeted radiotherapy of human melanoma xenografts: treatment of micrometastases. Cancer Res. 1990;50(10):2963–7. [PubMed] [Google Scholar]
  • 32.Kiess AP, Minn I, Vaidyanathan G, et al. (2S)-2-(3-(1-Carboxy-5-(4–211At-Astatobenzamido)Pentyl)Ureido)-Pentanedioic acid for PSMA-targeted α-particle radiopharmaceutical therapy. J Nucl Med. 2016;57(10):1569–75. 10.2967/jnumed.116.174300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Reilly RM, Georgiou CJ, Brown MK, Cai Z. Radiation nanomedicines for cancer treatment: a scientific journey and view of the landscape. EJNMMI Radiopharm Chem. 2024;9(1):37. 10.1186/s41181-024-00266-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Qin S, Yang Y, Zhang J, et al. Effective treatment of SSTR2-positive small cell lung cancer using (211)At-Containing targeted α-particle therapy agent which promotes endogenous antitumor immune response. Mol Pharm. 2023;20(11):5543–53. 10.1021/acs.molpharmaceut.3c00427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Luna-Gutiérrez M, Cruz-Nova P, Jiménez-Mancilla N, et al. Synthesis and evaluation of (177)Lu-DOTA-PD-L1-i and (225)Ac-HEHA-PD-L1-i as potential radiopharmaceuticals for tumor microenvironment-targeted radiotherapy. Int J Mol Sci. 2023;24(15):12382. 10.3390/ijms241512382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Huang X, Kaneda-Nakashima K, Kadonaga Y, et al. Astatine-211-Labeled gold nanoparticles for targeted alpha-particle therapy via intravenous injection. Pharmaceutics. 2022;14(12):2705. 10.3390/pharmaceutics14122705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Liu Y, Zhou Z, Feng Y, et al. Gold nanostars: A novel platform for developing (211)At-Labeled agents for targeted alpha-particle therapy. Int J Nanomedicine. 2021;16:7297–305. 10.2147/ijn.S327577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Falzone N, Ackerman NL, Rosales LF, et al. Dosimetric evaluation of radionuclides for VCAM-1-targeted radionuclide therapy of early brain metastases. Theranostics. 2018;8(1):292–303. 10.7150/thno.22217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Iizuka Y, Manabe Y, Ooe K, et al. Exploring a nuclear-selective radioisotope delivery system for efficient targeted alpha therapy. Int J Mol Sci. 2023;24(11):9593. 10.3390/ijms24119593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Lejeune P, Cruciani V, Berg-Larsen A, et al. Immunostimulatory effects of targeted thorium-227 conjugates as single agent and in combination with anti-PD-L1 therapy. J Immunother Cancer. 2021;9(10):e002387. 10.1136/jitc-2021-002387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Ertveldt T, Krasniqi A, Ceuppens H, et al. Targeted α-therapy using (225)Ac radiolabeled single-domain antibodies induces antigen-specific immune responses and instills immunomodulation both systemically and at the tumor microenvironment. J Nucl Med. 2023;64(5):751–8. 10.2967/jnumed.122.264752. [DOI] [PubMed] [Google Scholar]
  • 42.Ryan CJ, Saylor PJ, Everly JJ, Sartor O. Bone-targeting radiopharmaceuticals for the treatment of bone-metastatic castration-resistant prostate cancer: exploring the implications of new data. Oncologist. 2014;19(10):1012–8. 10.1634/theoncologist.2013-0472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Weller M, Albert N L, Galldiks N, et al. Targeted radionuclide therapy for gliomas: emerging clinical trial landscape. Neuro Oncol 2024; 26(Supplement_9):S208-S214. 10.1093/neuonc/noae125. [DOI] [PMC free article] [PubMed]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (DOC 23685 kb) (23.1MB, doc)
Supplementary file2 (PNG 161 kb) (161.7KB, png)

Data Availability Statement

Original data are available on request.

Articles from European Journal of Nuclear Medicine and Molecular Imaging are provided here courtesy of Springer

RESOURCES