Recent advances in the development of 225Ac- and 211At-labeled radioligands for radiotheranostics

Radiotheranostics utilizes a set of radioligands incorporating diagnostic or therapeutic radionuclides to achieve both diagnosis and therapy. Imaging probes using diagnostic radionuclides have been used for systemic cancer imaging. Integration of therapeutic radionuclides into the imaging probes serves as potent agents for radionuclide therapy. Among them, targeted alpha therapy (TAT) is a promising next-generation cancer therapy. The α-particles emitted by the radioligands used in TAT result in a high linear energy transfer over a short range, inducing substantial damage to nearby cells surrounding the binding site. Therefore, the key to successful cancer treatment with minimal side effects by TAT depends on the selective delivery of radioligands to their targets. Recently, TAT agents targeting biomolecules highly expressed in various cancer cells, such as sodium/iodide symporter, norepinephrine transporter, somatostatin receptor, αvβ3 integrin, prostate-specific membrane antigen, fibroblast-activation protein, and human epidermal growth factor receptor 2 have been developed and have made remarkable progress toward clinical application. In this review, we focus on two radionuclides, 225Ac and 211At, which are expected to have a wide range of applications in TAT. We also introduce recent fundamental and clinical studies of radiopharmaceuticals labeled with these radionuclides. Graphical abstract


Introduction
Radiotheranostics is a promising medical technology that uses a set of radioligands incorporating diagnostic or therapeutic radionuclides to achieve both diagnosis and therapy.For instance, by incorporating diagnostic radionuclides into cancer targeting agents, imaging diagnosis can provide information about the presence of targets and the accessibility of the agents.Subsequently, the introduction of therapeutic radionuclides into these imaging probes holds the potential to enable precise radionuclide therapy [1][2][3].Among them, targeted alpha therapy (TAT) is a cancer treatment approach that uses tumor-homing agents with α-particleemitting radionuclides (α-emitters) [4,5].The α-particles emitted from the TAT agents exhibit a constrained tissue range, usually affecting only a few number of cells (50-100 μm), enabling the specific irradiation of the target cancer cells.Moreover, α-particles possess a high linear energy transfer (LET) ranging from 50 to 230 keV μm −1 [6], enabling them highly effective in inducing cell death, primarily through the induction of double-strand breaks in DNA [7].Therefore, TAT is expected to be a precise therapy that can regress cancer cells while protecting healthy tissues.TAT is expected to revolutionize cancer treatment, by bringing a novel perspective to late-stage cancer, as treatment options are limited, and contributing to major advances in the field of cancer treatment [8].
Several useful α-emitters, including 223 Ra, 225 Ac, and 211 At, are currently used in clinical treatment modalities and clinical trials [9]. 223Ra has a half-life of 11.4 days, and its ionic form, [ 223 Ra]Ra 2+ , is clinically employed in treating bone metastatic prostate cancer as a commercially available radiopharmaceutical named Xofigo [10]. 223Ra has a reasonably long half-life and is anticipated to be a valuable nuclide for TAT.Nevertheless, developing an appropriate stable chelator of 223 Ra for clinical applications is presently a challenging obstacle, impeding progress in the development of radiotracers for various targets. 225Ac has multiple α-particles with high energy (5.8-7.1 MeV) and sufficient half-life (t 1/2 = 9.9 days) for high therapeutic efficacy (Fig. 1) [11].Furthermore, it can establish stable complexes by binding to ligands like 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-acetic acid (DOTA), enabling its use as versatile bifunctional agents within any cancer-targeted molecules [12].Hence, 225 Ac is recognized as one of the most effective α-emitters for cancer therapy.The release of radioactivity from target tumor tissues owing to the desorption of the daughter nuclides of 225 Ac from the chelator is a problem that needs to be addressed.In addition, the restricted availability of 229 Th, which serves as the primary source of 225 Ac, hinders the global distribution of this radionuclide.Recently, 211 At has also been considered as a promising α-emitter for TAT [13].The relatively short half-life of 211 At (t 1/2 = 7.2 h) gives rise to various issues, including the challenges of guaranteeing an ample supply of therapeutic doses and facilitating the distribution of 211 At from manufacturing facilities to medical institutions where it is employed.Conversely, 211 At possesses distinct advantages over other α-emitters with longer half-lives, like 225 Ac. 211 At is produced by the nuclear reaction of 209 Bi(α, 2n) 211 At using a cyclotron from 209 Bi, which is relatively easy to obtain. 211At decays with 5.87MeV of α-emission to transform into 207 Bi, which subsequently decays via electron capture (EC) into stable 207 Pb (Fig. 2).In the second branched decay, 211 At can also undergo EC decay to form 211 Pb, followed by the emission of α-particle (7.45 MeV) to produce 207 Pb.In other words, 211 At emits 100% α-particles in decay, and unlike the decay of 225 Ac, longlived α-particle-emitting daughter nuclides are not produced [14].Similar to its cognate halogen atoms, 123/131 I, 211 At forms biologically stable molecules that covalently binds to benzene rings and neopentyl groups [15,16].It is expected to serve as a versatile bifunctional molecules that binds to cancer-targeting molecules, such as 225 Aclabeled agents, to develop diverse TAT agents.Another major advantage is the ease of imaging the biodistribution of 211 At-labeled compounds by detecting 211 Po-derived X-rays with a gamma camera or single photon emission computed tomography (SPECT) [17].
In this context, a recent surge has been observed in research focused on the development and clinical applications of new drugs labeled with 225 Ac and 211 At.This review focuses on the recent advances in radiopharmaceuticals labeled with 211 At and 225 Ac and offers a comprehensive overview of their synthesis, biological evaluation, and clinical applications.

Radiolabeled compounds for thyroid cancers
Thyroid cancer therapy is based on surgery followed by radioiodine therapy.Radioiodine treatment with radioiodine diagnosis was first conducted by Dr. Hertz in 1942 [18][19][20], marking the beginning of radiotheranostics.Radioiodine diagnosis and treatment are based on iodine uptake into differentiated thyroid cancer cells by the sodium/ iodide symporter (NIS), and this theranostic strategy is applicable for NIS-expressing cancers including metastatic regions.Radioiodine has been used to treat thyroid diseases for more than 80 years.However, some patients with multiple metastases are refractory to repetitive radioiodine ( 131 I) treatment despite sufficient iodine uptake in targeted regions [21,22].A more effective strategy is required to treat radioactivity-refractory cancer in such cases.

211
At, a halogen element with chemical properties similar to those of iodine, has been gained attention as an α-emitter.
[ 211 At]Astatide also accumulates in cancer cells mediated by NIS [23,24].These characteristics are similar to those of [ 131   I]NaI [26].Furthermore, therapeutic experiments using NIS-expressing tumor-bearing mice demonstrated complete primary tumor eradication with no recurrence during 1-year follow-up [27].The major features of 211 At are a potent therapeutic effect and an extremely short range that reduces radiation exposure to surrounding people, enabling outpatient treatment without requiring admission to a dedicated hospital room.Therefore, Phase I trials are currently underway in Japan.[ 211 At]NaAt is being investigated in patients with differentiated thyroid cancer at Osaka University Hospital to establish the recommended dose for Phase II trials (NCT05275946).

Radiolabeled compounds for norepinephrine (NE) transporter-expressing cancers
Neuroblastoma is a pediatric cancer originating from the sympathetic nervous system, often characterized by metastasis and recurrence, and is often inoperable in many instances [28,29].Pheochromocytomas and paragangliomas are rare neuroendocrine tumors (NETs) associated with a relatively high incidence of local invasion or metastasis, rendering some cases unsuitable for surgical intervention [30,31].Most of these tumors express high levels of NE transporters [28,32].Because m-[ 123 I]iodobenzylguanidine ([ 123 I]MIBG) (Fig. 3a) is a substrate for the NE transporter, SPECT imaging with this radioligand has been used to diagnose these tumors such as neuroblastomas [33].The m-[ 131 I]iodobenzylguanidine ([ 131 I]MIBG) (Fig. 3b), wherein the β − -emitter 131 I replaces 123 I, has been clinically utilized as an effective therapeutic radioligand for tumors expressing the NE transporter.Response rates of over 30% have been observed when administered as a single agent [34,35].Nevertheless, its effect is frequently short-lived because the β − -particles from [ 131 I] MIBG may not be optimal for effectively eradicating isolated cells or small cell clusters due to their extended path lengths [36,37].Hence, m-[ 211 At]astatobenzylguanidine ([ 211 At]MABG), where the meta-position 131 I of [ 131 I] MIBG is substituted with 211 At (Fig. 3c), α-emitter capable of focusing high energy within a more confined area, gained attention.[ 211 At]MABG demonstrated the physicochemical properties similar to those of [ 131 I]MIBG and specific uptake by neuroblastoma cells in vitro [38].[ 211 At] MABG showed a similar disposition to [ 131 I]MIBG in SK-N-SH tumor-bearing mice but with higher accumulation in the tumor and heart [39].Accordingly, [ 211 At]MABG was anticipated to pave the way for a novel TAT that could surpass existing treatments for NE transporter-expressing tumors.[ 211 At]MABG showed notably higher cytotoxicity in the non-exposed group than in spheroids consisting of SK-N-BE(2c) in neuroblastoma from 0.48 kBq/ mL [40].The maximum tolerated dose of [ 211 At]MABG ranged from 51.8 to 66.7 MBq/kg in a mouse model of disseminated neuroblastoma transplanted with cells that overexpress the NE transporter.The results indicated that a single dose (66.7 MBq) or four divided doses (16.6 MBq) resulted in notable tumor regression effects and extended survival [41].[ 211 At]MABG has also shown remarkable therapeutic efficacy in treating malignant pheochromatoma.The administration of [ 211 At]MABG (0.56 MBq) to rat pheochromatoma PC12 tumor-bearing mice resulted in a notable tumor regression effect, with tumors being 53 times smaller after 21 days than those in the control group (relative tumor volumes of 509% and 9.6% when compared to control, respectively) [42].Analysis of mRNA expression in response to [ 211 At]MABG indicated that change in the p53-p21-dependent cell cycle checkpoint notably inhibits the growth of PC12 cells [43].Evaluation of the acute radiation-related toxicity of [ 211 At]MABG in ICR mice revealed that a maximum tolerated dose of 3.3 MBq.Despite the high absorbed doses in numerous organs, such as the thyroid, heart, stomach, and adrenal glands, no unexpected severe toxic effects were observed in the mice [44].Fukushima Medical University Hospital has commenced a phase I dose-escalation study of [ 211 At] MABG in patients diagnosed with malignant pheochromocytoma or paraganglioma (jRCT2021220012) [13].On the other hand, the uptake of [ 211 At]MABG by the non-target organic cation transporter 3 poses a risk of potential side effects in normal tissues, and, therefore, warrants careful consideration in the treatment process [45].The combination of histone deacetylase inhibitors such as Vorinostat and [ 211 At]MABG exhibits a synergistic neuroanticancer effect on neuroblastoma.This effect may be attributed to reduced expression of DNA damage repair proteins and increased expression of NE transporter proteins [46,47].Additional basic and clinical studies of [ 211 At]MABG are anticipated in the future, including investigations to confirm whether combination therapy with other agents can enhance its therapeutic effectiveness.

Radiolabeled octreotide analogs with high affinity for somatostatin receptors
NETs are neoplasms arising from endocrine cells primarily found in the gastrointestinal tract, pancreas, lungs, and other tissues [48].NETs typically demonstrate a highly differentiated, low-proliferative character and often require surgical intervention for a complete cure [49].However, in some cases, they may be unresectable during detection and chemotherapy tends to be less effective.Somatostatin receptors (SSTRs), particularly SSTR2, are highly expressed in NETs.Consequently, 68 Ga-labeled octreotide derivatives of cyclic peptides, including [ 68 Ga]Ga-DOTATATE (Fig. 4a) and [ 68 Ga]Ga-DOTATOC (Fig. 4b), have been employed for positron emission tomography (PET) diagnosis of tumors expressing SSTRs and for providing prognostic information  [50].For radionuclide therapy, the clinical application of [ 177 Lu]Lu-DOTATATE (Fig. 4c) radiolabeled with a β −emitter 177 Lu with specific affinity for SSTR2, has proven efficacious in the treatment of metastatic and unresectable NETs [51].However, certain tumors demonstrate resistance or recurrence when subjected to this therapeutic approach [52].The lower LET of β − -particles from 177 Lu (~ 0.2 keV/ μm) compared to α-particles is associated with their primary mechanism of inducing single-strand DNA breaks, which may explain their limited therapeutic effectiveness.Therefore, in anticipation of the efficacy of NETs with TAT, fundamental and clinical studies on the therapeutic effects on NETs of octreotide derivatives labeled with 225 Ac, which can form stable complexes with DOTA as well as 177 Lu, were subsequently conducted [8,53].
Cyclic peptides DOTATOC and DOTATATE, known for their high affinity for SSTR2 and labeled with radiometals ( 68 Ga for diagnostic purposes and 90 Y and 177 Lu for therapy), are already employed in clinical practice [54].Accordingly, the initial preclinical investigations focused on DOTA peptides labeled with 225 Ac (Fig. 4c, d).[ 225 Ac]Ac-DOTATOC (12-20 kBq) suppressed the growth of NETs inoculated in mice more effectively than [ 177 Lu]Lu-DOTATOC (450-1000 kBq), and no toxicity was observed up to 20 kBq [55].A single administration of [ 225 Ac]Ac-DOTATATE (144-148 kBq) resulted in a remarkable tumor growth delay and extended the time to the experimental endpoint in SSTR2-positive lung cancer cell-transplanted mice compared to that of the control group [56].Both [ 225 Ac]Ac-DOTATOC and [ 225 Ac] Ac-DOTATATE exhibited nephrotoxicity at high doses (30 and 111 kBq, respectively), which was attributed to their substantial renal accumulation.[ 225 Ac]Ac-MACROPATATE (Fig. 4e) showed better serum stability with a chelator different from that of [ 225 Ac]Ac-DOTATATE.However, its antitumor effect was lower than that of [ 225 Ac]Ac-DOTA-TATE, its accumulation in the liver and kidney was higher, and its superiority over existing radioligands has not been verified [57].A preclinical study on lung cancer-bearing mice treated with 211 At-labeled octreotide ([ 211 At]SAB-Oct) has also been reported.Significant tumor regression was observed after 370 kBq administration compared with that of control group.A total of 1110 kBq administered in  5).This led to partial remission and stability, with no progression or death in the 8-month follow-up [64].
Recently, [ 225 Ac]Ac-DOTATATE therapy improved overall survival of 91 patients with SSTR-expressing NETs.Treatment-related toxicity was minimal, suggesting that overall survival could be improved even in patients refractory to previous [ 177 Lu]Lu-DOTATATE therapy [65].Several other case reports have also highlighted the clinical advantages of [ 225 Ac]Ac-DOTATATE, including complete remission in multiple patients [66][67][68][69].Although persistent concerns regarding nephrotoxicity are likely to drive the development of new 225 Ac-labeled SSTR-targeted agents, TAT with [ 225 Ac]Ac-DOTATATE has great potential as a potent treatment for NETs in clinical practice.In basic research, complexes with β − -emitters, such as [ 186 Re]Re-MAG3, [ 90 Y]Y-DOTA, and [ 177 Lu]Lu-DOTA, conjugated bisphosphonate compounds, which are carriers to bone lesions, were developed for the palliation of bone metastases (Fig. 6a-c) [74][75][76].These compounds showed high uptake in bone and low uptake in non-target tissues, indicating that the drug design concept is useful for bone-seeking radiopharmaceuticals.Moreover, the replacement of radionuclides for therapy to ones for imaging could adopt ideal radiotheranostics because diagnostic and therapeutic radiopharmaceuticals could show equivalent pharmacokinetics [77][78][79].

Radiolabeled RGD peptides with high affinity for α v β 3 integrin-expressing cancers
RGD peptides contain arginine-glycine-aspartic acid (RGD) sequence.RGD peptides have a high affinity for α v β 3 integrin, which is a heterodimeric transmembrane receptor for cell adhesion molecule [81].α v β 3 integrin, one of the integrin subtypes, regulates angiogenesis and is related to tumor development [82].As the α v β 3 integrin is highly expressed on endothelial cells in neovascularity and some types of cancer cells, RGD peptides have been used as carriers to cancer tissue [83,84].
Radiolabeled RGD peptides have been enthusiastically developed for cancer imaging and therapy in nuclear medicine [85][86][87].Utilization of the RGD tripeptide had been hindered by its short half-life in the blood and insufficient affinity.To overcome the limitation, structural modifications involving the incorporating an additional two amino acids, utilizing D-amino acid residues, and cyclizing the peptides have been implemented to improve the affinity for α v β 3 integrin and its bioavailability.Notably, c(RGDfK) and c(RGDyK) have emerged as fundamental constructs for developing radiolabeled RGD peptides [88].Furthermore, multimeric RGD peptides, such as dimer and tetramer, have been investigated to enhance affinity for α v β 3 integrin [89].Radiolabeled RGD peptides were explored for imaging purposes to determine α v β 3 integrin expression.Subsequently, The first report on radiotheranostics application with RGD peptide in a patient with papillary thyroid carcinoma was published in 2018 [90].This report described a combination of [ 68  Radiotheranostics is generally performed by introducing diagnostic and therapeutic radionuclides with similar chemical properties into the same precursor.Therefore, the combinations of radionuclides for radiotheranostics are limited.To overcome the limitation, multiradionuclide radiotheranostics with a combination of [ 67 Ga]Ga-DOTAc[RGDf(4-I)K], in which 67 Ga is an alternative radionuclide to 68 Ga, and Ga-DOTA-[ 211 At]c[RGDf(4-At)K] were developed by introducing a halogen introduction site and a metal complex in a molecule (Fig. 9c, d) [92].To increase tumor accumulation and retention of 211 At-labeled RGD peptide, an albumin-binding moiety (ABM) was introduced (Fig. 9e).Ga-DOTA-K([ 211 At]APBA)-c(RGDfK) with ABM delayed blood clearance, increased tumor accumulation compared to compounds without ABM, and showed strong therapeutic effects in tumor-bearing mice [93].Clinical applications of TAT using RGD peptides are expected in the future.
PSMA, a cell surface enzyme consisting of 750 amino acids with a molecular weight of 87 kDa, is predominantly expressed in prostate epithelial cells.PSMA is not released into the blood and is overexpressed in prostate cancer, exhibiting a progressive increase in its expression with higher tumor grades [96,97].As the PSMA expression level is a significant indicator for predicting disease outcomes in patients with prostate cancer [98], PSMA could be an appropriate target for radiotheranostics.
Almost all radiolabeled PSMA ligands have recently been shown to possess a Glu-urea-Lys pharmacophore.Moreover, PSMA ligands with a lipophilic linker increased the binding affinity for PSMA due to a hydrophobic pocket adjacent to the pharmacophore [99,100].In 2012, [ 68 Ga]Ga-PSMA-11 (Fig. 10a) was reported to exhibit high PSMAspecific internalization in prostate cancer cells and excellent PET images [101].Meanwhile, the HBED-CC chelate for 68 Ga in PSMA-11 does not coordinate with therapeutic radiometals such as 177 Lu.Subsequently, PSMA-617, a pharmacophore Glu-urea-Lys conjugated DOTA chelator (Fig. 10b) was developed via a lipophilic linker optimized for properties such as length, polarity, size, flexibility, and the presence of aromatic groups [102].
About TAT-targeting PSMA, the surprising therapeutic effects of [ 225 Ac]Ac-PSMA-617 were reported in clinical studies in 2016 (Fig. 11) [103].The initial clinical encounter  [90] with [ 225 Ac]Ac-PSMA-617 revealed encouraging antitumor efficacy.The duration of response is 10-15 months with complete remission in approximately 10% of patients, while some patients have sustained relapse-free survival [104].In basic research, superior 111 In/ 225 Ac-labeled compounds targeting PSMA have been developed for cancer

Radiolabeled fibroblast-activation protein inhibitors (FAPIs)
The tumor microenvironment is composed of stromal components, with cancer-associated fibroblasts (CAFs) representing the predominant component of the tumor stroma [110].Cancer cells secrete growth factors that induce the transformation of fibroblasts into CAFs.This activation process leads to high expression of CAF markers such as  fibroblast-activating protein (FAP), a type II transmembrane protease known to facilitate tumor growth and metastasis.Moreover, FAP is prominently expressed on the cell surface of activated fibroblasts, as observed in over 90% of epithelial cancers, whereas it is absent in normal adult tissues.Consequently, FAP has been identified as a suitable target for imaging and therapy of various types of tumors [111].Recently, several clinical trials of 68 Ga-or 18 F-labeled FAP inhibitors (FAPIs) PET imaging have been performed [112].These studies with superior PET images indicate that radiolabeled FAPIs could be an important target for cancer theranostics.
[ 225 Ac]Ac-FAPI-04 (Fig. 13a) was first reported in 2020 [113].[ 225 Ac]Ac-FAPI-04 significantly inhibited tumor growth in the PANC-1 tumor-bearing mice compared with that in control mice, without a significant body weight loss.Meanwhile, the clearance of [ 225 Ac]Ac-FAPI-04 from the tumor appeared to be too rapid for the physical half-life of 225 Ac.To improve the tumor retention of the FAPI compounds, FAPI-46 was developed to show a better retention than FAPI-04 [114].Thus, [ 225 Ac]Ac-FAPI-46 was reported in 2022 (Fig. 13b) [115].However, the therapeutic effects of [ 225 Ac]Ac-FAPI-46 were limited.In other words, the tumor-suppressive effects were not significant compared to those in the control group.The improvement in retention was likely insufficient, and the biological halflife of FAPI-46 was too short for the physical half-life of 225 Ac.
These studies indicate that radiotheranostics containing TAT-targeting FAP in the cancer stroma is effective.Although the detailed therapeutic mechanism is not clear, it could be a new cancer therapeutic strategy in combination with other therapies directly targeting cancer cells.

Radiolabeled antibodies and their fragments to cancer cell membrane antigens
PET diagnosis using antibodies (also called ImmunoPET) has been developed as a target specific diagnostic tool for various types of cancer [118].Radiolabeled antibodies could be also applied for radionuclide therapy due to excellent target specificity of antibodies.The anti-CD20 monoclonal antibodies [ 131 I]I-tositumomab (Bexxar ® ) and [ 90 Y]Y-ibritumomab thiuxetan (Zevalin ® ), labeled with β − -emitters, have been used to treat non-Hodgkin's lymphoma [119].Immunoglobulin G (IgG) antibodies (M w = 150 kDa) have a very high affinity and specificity for their targets (Fig. 15a), making them suitable vectors for TAT [120].In addition, owing to their long half-life in blood, α-emitters with relatively long half-lives, such as 225 Ac, are expected to deliver effective antitumor effects.Therefore, IgG-based TAT agents have been primarily developed as 225 Ac-labeled agents.Fundamental studies were conducted in cells and mice using 225 Ac-labeled IgG antibodies.Their targets include the human epidermal growth factor receptor 2 (HER2) [121,122], epidermal growth factor receptor (EGFR) [123], PSMA [124], CD46 [125], CD33 [126,127], CD20 [128], Carbonic Anhydrase IX [129], Podoplanin [130], and carcinoembryonic antigen [131]. 211At-labeled antibodies (and antibody fragments) were also evaluated for CD38 [132], CD123 [133], CD33 [134], CD45 [135], and membrane phosphate transporter protein (NaPi2b).[136] Among these targets, clinical trials have been reported the treatment of acute myeloid leukemia targeting CD33 and ovarian cancer  Variable fragments of heavy chain antibodies (VHH), smaller in size (12-15 kDa) and less immunogenic than IgG (Fig. 15b), are rapidly cleared from the blood and non-target tissues while maintaining affinity and specificity.Recombinant VHHs can be produced in bulk to reduce costs [137].This has driven research on VHHs for TAT, including radioligands, not only 225 Ac but also 211 At, owing to their shorter blood half-life.Preclinical studies on 211 At-or 225 Ac-labeled VHH for HER2 [138,139], CD20 [140], and 5T2MM idiotypes [141] have also been reported.In biodistribution studies in mice, these VHH-based radioligands reached a plateau in the tumor tissue within about 3-6 h, and showed significantly higher therapeutic efficacy than the non-treated groups.Ertveldt et al. reported that 225 Ac-anti-CD20 VHH induced systemic antitumor immune responses, suggesting that combination therapy with TAT and tumor immunotherapy may be a promising new cancer treatment tool [140].However, nephrotoxicity based on the physiological accumulation of VHH has been observed, and caution should be exercised in future clinical applications.

Radiolabeled nanoparticles for tumor microenvironment
Nanoparticles have gained attention as drug delivery carriers.Nanocarriers are used in nuclear medicine to develop nanoradiopharmaceuticals labeled with γ-or positronemitter for diagnosis and α-or β − -emitter for therapy [142]. 225Ac is a promising α-emitter for TAT using nanoparticles since its relatively long half-life (9.9 days) is suitable for the biodistribution of nanoparticles retained in tumors.
Liposomes are well-known carriers of active agents, including radiolabeled compounds.Sofou et al. successfully loaded 225 Ac into liposomes with a high encapsulation efficiency, whereas 213 Bi, the α-particle-emitting daughter of 225 Ac, was poorly retained in the liposomes [143].Maintaining the α-particle-emitting daughters within liposomes during delivery to tumors is important as the cell-killing efficacy of 225 Ac is partially derived from α-particles emitted from three α-particle-emitting daughters ( 221 Fr, 217 At, and 213 Bi) generated during 225 Ac decay (Fig. 1).However, some loss is unavoidable owing to the recoil effect associated with the emission of α-particles from daughters with a recoil distance of 80-90 nm.Increased retention of 213 Bi has been observed in liposomes with increased particle sizes [143] and in multivesicular liposomes [144].To enable the therapeutic use of 225 Ac-containing liposomes, encapsulation efficiency was improved by up to 73% using the active loading method [145].The 225 Ac-containing liposomes modified with antibodies or aptamers targeting PSMA show selective accumulation and cytotoxicity in PSMA-expressing cells [146].In addition, 225 Ac-labeled liposomes inhibited tumor growth in tumor-bearing mice [147].
Gold nanoparticles [148], LnPO 4 nanoparticles [149], and calcium core-shell particles [150] have also been reported as 225 Ac-labeled nanoparticles.Gold nanoparticles were labeled by chelating 225 Ac via the chelator DOTAGA, which was modified on the surface of the gold nanoparticles.Although daughters were not retained with gold nanoparticles owing to the alpha recoil effect, in vitro and in vivo therapeutic effects were observed.However, LnPO 4 nanoparticles and calcium core-shell particles doped with 225 Ac in the core of the nanoparticles were designed to retain 225 Ac as well as α-particle-emitting daughters.Both nanoparticles exhibited high in vivo stability and biodistribution of 213 Bi, the last α-particle-emitting daughter, was similar to that of 225 Ac.
Few studies have used 211 At-labeled nanoparticles due to the short half-life (7.2 h) of 211 At.However, 211 At-labeled gold nanoparticles have been developed as 211 At can be adsorbed onto gold nanoparticles by simple mixing.The intratumoral injection of 211 At-labeled gold nanoparticles inhibited tumor growth [151].The therapeutic effects were dependent on the size of gold nanoparticles; those with a diameter of 5 nm showed the strongest therapeutic effects among those with diameters of 5, 13, 30, and 120 nm.Intravenous injection was also evaluated; 211 At-labeled gold nanoparticles exhibited potent therapeutic effects in a PANC-1 xenograft model [152].

Radiolabeled compounds with albumin-binding moiety (ABM) for improved pharmacokinetics and tumor targeting
Albumin is the most abundant protein in the body with a biological half-life of 19 days.Albumin contains several distinct binding pockets and is a carrier for endogenous and exogenous compounds such as lipids, hormones, metal ions, and lipophilic drugs.In nuclear imaging, fast clearance of radiolabeled compounds from the blood is generally preferred to achieve a high-tumor-to-blood ratio, which is important for imaging.However, rapid blood clearance can limit tumor uptake, making it difficult to use radiolabeled compounds for therapeutic applications.To overcome these problems, low-molecular-weight albumin-binding molecules such as 4-(4-iodophenyl)butyric acid and Evans blue derivatives have been used for therapeutic applications (Fig. 16) [153,154].These albumin binders exhibit non-covalent, reversible interactions with albumin, which extend the in vivo blood circulation time of the radiotracers.Since the dissociation constants of radiotracers against albumin in the low micromolar range are higher than those against targeted receptors in the nanomolar range, increased accumulation in tumors can be achieved by conjugating ABM to conventional radiotracers containing a tumor-targeting moiety.
Radiotracers containing ABM have been developed as theranostic probes targeting tumor-expressing molecules such as PSMA, SSTR, α v β 3 integrin, folate receptor, glucagon-like peptide-1 receptor and bone [105,[155][156][157].There are many reports on the use of DOTA derivatives as chelators for radiometals such as 67 Ga, 68 Ga, and 111 In for diagnostic imaging and 90 Y and 177 Lu for therapeutic applications.
DOTA derivatives used as chelators of 225 Ac and 225 Aclabeled probes with ABM targeting PSMA (SibuDAB, Fig. 17a) showed increased blood retention, high-tumor accumulation, and potent therapeutic efficacy in PSMAexpressing tumor-bearing mice [158].The 18-membered macrocycle macropa derivatives have also been described as chelators for 225 Ac, allowing rapid complexation at room temperature [159].[ 225 Ac]Ac-macropa conjugated compounds with one or two albumin-and PSMA-targeting moieties (mcp-M-alb-PSMA and mcp-d-alb-PSMA, Fig. 17b, c) prolonged the blood circulation time, specifically and highly accumulated in the tumor, and inhibited tumor growth with DNA double-strand break formation [160].
4-(4-Astatophenyl)butyric acid (APBA), in which the iodine in 4-(4-iodophenyl)butyric acid is replaced with astatine, also functions as an ABM, as described in the RGD peptide section [93].Although only a few reports are present on 211 At-labeled compounds containing ABM owing to the short half-life of 211 At, APBA can be applied to other probes with different targeting moieties, which may facilitate the development of 211 At-labeled compounds containing ABM.The affinity of the probes for albumin was closely related to the kinetic profile of tumor uptake [161].Lysine-based albumin binders with lower albumin-binding affinities showed higher calculated areas under the curve (AUC) in the tumors among the probes exhibiting albumin-binding affinities from 1.8 to 50 μM.To decrease uptake in normal organs is advantageous for probes containing ABM, such as the kidneys.AUC for the kidney was not affected by the binding affinity of the probes.These results are valuable for designing novel probes containing ABM and will facilitate the development of probes useful for endoradionuclide therapy.

Conclusion
TAT is a promising treatment in oncology owing to its high cytotoxicity in cancer cells.For TAT, developing probes that deliver α-emitters to the tumor tissues is important.Recently, various probes have been designed targeting molecules specifically expressed in tumors, such as α v β 3 integrin, PSMA, FAP, and SSTR.In preclinical studies using tumorbearing mice, various probes have exhibited high therapeutic efficacy without serious side effects.Clinical trials are also being conducted, including two in Japan, using [ 211 At]NaAt and [ 211 At]MABG.The endoradionuclide therapy using α-emitters is expected to be approved and contribute to the treatment of many cancer patients in the near future.
Funding Open Access funding provided by Kanazawa University.

Fig. 1
Fig. 1 Decay scheme of 225 Ac I]iodide, suggesting that [ 211 At]astatide is a possible alternative radionuclide to [ 131 I]iodide in NIS-based endoradiotherapy.A toxicity study demonstrated no severe side effects in normal mice intravenously administered with [ 211 At]NaAt solutions up to 50 MBq/kg [25].In addition, the [ 211 At]NaAt induced more DNA double-strand breaks and decreased colony formation than [ 131 I]NaI and a stronger tumor-growth suppression was observed in mice injected with 0.4 and 0.8 MBq of [ 211 At]NaAt than those injected with 1.0 MBq of [

Fig. 4
Fig. 4 (continued) Many bone-seeking agents with β − -emitter for palliation of bone metastases, such as [ 89 Sr]SrCl 2 and [ 153 Sm]Sm-EDTMP, have been developed for a long time[70,71].However, bone-seeking radiopharmaceuticals with β − -emitters do not prolong the overall survival in patients.Meanwhile, [ 223 Ra]RaCl 2 significantly prolonged the overall survival of castration-resistant prostate cancer patients with bone metastases in a phase III study[72].Following the results of the phase III study, [ 223 Ra]RaCl 2 was approved by the U.S. Food and Drug Administration (FDA) as the first therapeutic radiopharmaceutical with an α-emitter.Although [ 223 Ra] RaCl 2 and the bone scintigraphy agents do not have precisely

Fig. 15
Fig.15 Structures and properties of immunoglobulin G (IgG) antibodies and variable fragments of heavy chain antibodies (VHH).CH constant heavy; VH variable heavy; VL variable light