BACKGROUND: Astatine-211 has attained significant interest in the recent times as a promising radioisotope for targeted alpha therapy (TAT) of cancer. In this study, we report the production of 211At via 209Bi (α, 2n) 211At reaction in a cyclotron and development of a facile radiochemical separation procedure to isolate 211At for formulation of nanoradiopharmaceuticals.
METHODS: Natural bismuth oxide target in pelletized form wrapped in Al foil was irradiated with 30 MeV α-beam in an AVF cyclotron. The irradiated target was dissolved in 2 M HNO3 followed by selective precipitation of Bi as Bi(OH)3 under alkaline condition. The radiochemically separated 211At was used for labeling cyclic RGD peptide conjugated gold nanoparticles (Au-RGD NPs) by surface adsorption. The radiochemical stability of 211At-Au-RGD NPs was evaluated in phosphate buffered saline (PBS) and human serum media.
RESULTS: The batch yield of 211At at the end of irradiation was ∼6 MBq.μA-1.h-1. After radiochemical separation, ∼80 % of 211At could be retrieved with >99.9 % radionuclidic purity. Au-RGD NPs (particle size 8.4±0.8 nm) could be labeled with 211At with >99 % radiolabeling yield. The radiolabeled nanoparticles retained their integrity in PBS and human serum media over a period of 21 h.
CONCLUSIONS: The present strategy simplifies 211At production in terms of purification and would increase affordable access to this radioisotope for TAT of cancer.

Cancer is one of the most complex and challenging human diseases, with rising incidences and cancer-related deaths despite improved diagnosis and personalized treatment options. Targeted alpha therapy (TαT) offers an exciting strategy emerging for cancer treatment which has proven effective even in patients with advanced metastatic disease that has become resistant to other treatments. Yet, in many cases, more sophisticated strategies are needed to stall disease progression and overcome resistance to TαT. The combination of two or more therapies which have historically been used as stand-alone treatments is an approach that has been pursued in recent years. This review aims to provide an overview on TαT and the four main pillars of therapeutic strategies in cancer management, namely external beam radiation therapy (EBRT), immunotherapy with checkpoint inhibitors (ICI), cytostatic chemotherapy (CCT), and brachytherapy (BT), and to discuss their potential use in combination with TαT. A brief description of each therapy is followed by a review of known biological aspects and state-of-the-art treatment practices. The emphasis, however, is given to the motivation for combination with TαT as well as the pre-clinical and clinical studies conducted to date.

BACKGROUND: Targeted alpha therapy (TAT) of somatostatin receptor-2 (SSTR2) positive neuroendocrine tumors (NETs) involving Ac-225 ([225Ac]Ac-DOTA-TATE) has previously demonstrated improved therapeutic efficacy over conventional beta particle-emitting peptide receptor radionuclide therapy agents. DOTA-TATE requires harsh radiolabeling conditions for chelation of [225Ac]Ac3+, which can limit the achievable molar activities and thus therapeutic efficacy of such TAT treatments. Macropa-TATE was recently highlighted as a potential alternative to DOTA-TATE, owing to the mild radiolabeling conditions and high affinity toward [225Ac]Ac3+; however, elevated liver and kidney uptake were noted as a major limitation and a suitable imaging radionuclide is yet to be reported, which will be required for patient dosimetry studies and assessment of therapeutic benefit. Previously, [155Tb]Tb-crown-TATE has shown highly effective imaging of NETs in preclinical SPECT/CT studies, with high tumor uptake and low non-target accumulation; these favourable properties and the versatile coordination behavior of the crown chelator may therefore show promise for combination with Ac-225 for TAT.
METHODS: Crown-TATE was labeled with Ac-225, and radiochemical yield was analyzed as the function of crown-TATE concentration. LogD7.4 was measured as the indication of hydrophilicity. Free [225Ac]Ac3+ release from [225Ac]Ac-crown-TATE in human serum was studied. Biodistribution studies of [225Ac]Ac-crown-TATE in mice bearing AR42J tumors was evaluated at 1, 4, 24, 48, and 120 h, and the absorbed dose to major organs calculated. Therapy-monitoring studies with AR42J tumor bearing mice were undertaken using 30 kBq and 55 kBq doses of [225Ac]Ac-crown-TATE and compared to controls treated with PBS or crown-TATE.
RESULTS: [225Ac]Ac-crown-TATE was successfully prepared with high molar activity (640 kBq/nmol), and characterized as a moderately hydrophilic radioligand (LogD7.4 = -1.355 ± 0.135). No release of bound Ac-225 was observed over 9 days in human serum. Biodistribution studies of [225Ac]Ac-crown-TATE showed good initial tumor uptake (11.1 ± 1.7% IA/g at 4 h) which was sustained up to 120 h p.i. (6.92 ± 2.03% IA/g). Dosimetry calculations showed the highest absorbed dose was delivered to the tumors. Therapy monitoring studies demonstrated significant (log-rank test, P < 0.005) improved survival in both treatment groups compared to controls.
CONCLUSIONS: This preclinical study demonstrated the therapeutic efficacy of [225Ac]Ac-crown-TATE for treatment of NETs, and highlights the potential of using crown chelator for stable chelation of Ac-225 under mild conditions.

The utilization of actinium-225 (225Ac) radionuclides in targeted alpha therapy for cancer was initially outlined in 1993. Over the past two decades, substantial research has been conducted, encompassing the establishment of 225Ac production methods, various preclinical investigations, and several clinical studies. Currently, there is a growing number of compounds labeled with 225Ac that are being developed and tested in clinical trials. In response to the increasing demand for this nuclide, production facilities are either being built or have already been established. This article offers a concise summary of the present state of clinical advancements in compounds labeled with 225Ac. It outlines various processes involved in the production and purification of 225Ac to cater to the growing demand for this radionuclide. The article examines the merits and drawbacks of different procedures, delves into preclinical trials, and discusses ongoing clinical trials.

Bismuth-213 is a radionuclide of interest for targeted alpha therapy and is supplied via a radiochemical generator system through the decay of 225Ac. Radionuclide generators employ longer lived \"parent\" radionuclides to routinely supply shorter-lived \"daughter\" radionuclides. The traditional 225Ac/213Bi radiochemical generator relies on an organic cation exchange resin where 225Ac binds to the resin and 213Bi is routinely eluted. These resins degrade when they absorb large doses of ionizing radiation (>1 × 106 Gy/mg), which has been observed when the loading activity of 225Ac exceeds 2.59*109 Bq (70 mCi). Herein we report the development of an electrochemical generator for the supply of 213Bi that has the potential to overcome this limitation. Bismuth-213 spontaneously electrodeposits onto nickel foils in 0.1 M hydrochloric acid at 70 °C. Using this method, we were able to plate an average of 73 ± 4 % of the 213Bi in solution and obtain a final 213Bi recovery of 65 ± 8 % in 0.1 M citrate pH 4.5 via reverse electrolysis using titanium as the cathode. The recovered 213Bi had an average radiochemical purity of >99.8 % and was successfully used to radiolabel DOTATATE with an average radiochemical yield of 85.1 % (not optimized).

BACKGROUND: Actinium-225 is one of the most promising radionuclides for targeted alpha therapy. Its limited availability significantly restricts clinical trials and potential applications of 225Ac-based radiopharmaceuticals.
METHODS: In this work, we examine the possibility of 225Ac production from the thermal neutron flux of a nuclear reactor. For this purpose, a target consisting of 1.4 mg of 226Ra(NO3)2 (T1/2 = 1600 years) and 115.5 mg of 90 % enriched, stable 157Gd2O3 was irradiated for 48 h in the Breazeale Nuclear Reactor with an average neutron flux of 1.7·1013 cm-2·s-1. Gadolinium-157 has one of the highest thermal neutron capture cross sections of 0.25 Mb, and its neutron capture results in emission of high-energy, prompt γ-photons. Emitted γ-photons interact with 226Ra to produce 225Ra according to the 226Ra(γ, n)225Ra reaction. Gadolinium debulking and separation of undesirable, co-produced 227Ac from 225Ra was achieved in one step by using 60 g of branched DGA resin. After 225Ac ingrowth from 225Ra (T1/2 = 14.8 d), 225Ac was extracted from the 226Ra and 225Ra fraction using 5 g of bDGA resin and then eluted using 5 mM HNO3.
RESULTS: Measured activity of 225Ac showed that 6(1) kBq or 0.16(3) μCi (1σ) of 225Ra was produced at the end of bombardment from 0.9 mg of 226Ra.
CONCLUSIONS: The developed 225Ac separation is a waste-free process which can be used to obtain pure 225Ac in a nuclear reactor.

OBJECTIVE: The radionuclide pair cerium-134/lanthanum-134 (134Ce/134La) was recently proposed as a suitable diagnostic counterpart for the therapeutic alpha-emitter actinium-225 (225Ac). The unique properties of 134Ce offer perspectives for developing innovative in vivo investigations that are not possible with 225Ac. In this work, 225Ac- and 134Ce-labelled tracers were directly compared using internalizing and slow-internalizing cancer models to evaluate their in vivo comparability, progeny meandering, and potential as a matched theranostic pair for clinical translation. Despite being an excellent chemical match, 134Ce/134La has limitations to the setting of quantitative positron emission tomography imaging.
METHODS: The precursor PSMA-617 and a macropa-based tetrazine-conjugate (mcp-PEG8-Tz) were radiolabelled with 225Ac or 134Ce and compared in vitro and in vivo using standard (radio)chemical methods. Employing biodistribution studies and positron emission tomography (PET) imaging in athymic nude mice, the radiolabelled PSMA-617 tracers were evaluated in a PC3/PIP (PC3 engineered to express a high level of prostate-specific membrane antigen) prostate cancer mouse model. The 225Ac and 134Ce-labelled mcp-PEG8-Tz were investigated in a BxPC-3 pancreatic tumour model harnessing the pretargeting strategy based on a trans-cyclooctene-modified 5B1 monoclonal antibody.
RESULTS: In vitro and in vivo studies with both 225Ac and 134Ce-labelled tracers led to comparable results, confirming the matching pharmacokinetics of this theranostic pair. However, PET imaging of the 134Ce-labelled precursors indicated that quantification is highly dependent on tracer internalization due to the redistribution of 134Ce\'s PET-compatible daughter 134La. Consequently, radiotracers based on internalizing vectors like PSMA-617 are suited for this theranostic pair, while slow-internalizing 225Ac-labelled tracers are not quantitatively represented by 134Ce PET imaging.
CONCLUSIONS: When employing slow-internalizing vectors, 134Ce might not be an ideal match for 225Ac due to the underestimation of tumour uptake caused by the in vivo redistribution of 134La. However, this same characteristic makes it possible to estimate the redistribution of 225Ac\'s progeny noninvasively. In future studies, this unique PET in vivo generator will further be harnessed to study tracer internalization, trafficking of receptors, and the progression of the tumour microenvironment.

Astatine-211 (211At) has emerged as a promising radionuclide for targeted alpha therapy of cancer by virtue of its favorable nuclear properties. However, the limited in vivo stability of 211At-labeled radiopharmaceuticals remains a major challenge. This review provides a comprehensive overview of the current strategies for 211At radiolabeling, including nucleophilic and electrophilic substitution reactions, as well as the recent advances in the development of novel bifunctional coupling agents and labeling approaches to enhance the stability of 211At-labeled compounds. The preclinical and clinical applications of 211At-labeled radiopharmaceuticals, including small molecules, peptides, and antibodies, are also discussed. Looking forward, the identification of new molecular targets, the optimization of 211At production and quality control methods, and the continued evaluation of 211At-labeled radiopharmaceuticals in preclinical and clinical settings will be the key to realizing the full potential of 211At-based targeted alpha therapy. With the growing interest and investment in this field, 211At-labeled radiopharmaceuticals are poised to play an increasingly important role in future cancer treatment.

BACKGROUND: A significant challenge in cancer therapy lies in eradicating hidden disseminated tumor cells. Within Nuclear Medicine, Targeted Alpha Therapy is a promising approach for cancer treatment tackling disseminated cancer. As tumor size decreases, alpha-particles gain prominence due to their high Linear Energy Transfer (LET) and short path length. Among alpha-particle emitters, 211At stands out with its 7.2 hour half-life and 100% alpha emission decay. However, optimizing the pharmacokinetics of radiopharmaceuticals with short lived radionuclides such as 211At is pivotal, and in this regard, pretargeting is a valuable tool. This method involves priming the tumor with a modified monoclonal antibody capable of binding both the tumor antigen and the radiolabeled carrier, termed the \"effector molecule. This smaller, faster-clearing molecule improves efficacy. Utilizing the Diels Alder click reaction between Tetrazine (Tz) and Trans-cyclooctene (TCO), the Tz-substituted effector molecule combines seamlessly with the TCO-modified antibody. This study aims to evaluate the in vivo biodistribution of two Poly-L-Lysine-based effector molecule sizes (10 and 21 kDa), labelled with 211At, and the in vitro binding of the most favorable polymer size, in order to optimize the pretargeted radioimmunotherapy with 211At.
RESULTS: In vivo results favor the smaller polymer\'s biodistribution pattern over the larger one, which accumulates in organs like the liver and spleen. This is especially evident when comparing the biodistribution of the smaller polymer to a directly labelled monoclonal antibody. The smaller variant also shows rapid and efficient binding to SKOV-3 cells preloaded with TCO-modified Trastuzumab in vitro, emphasizing its potential. Both polymer sizes showed equal or better in vivo stability of the astatine-carbon bond compared to a monoclonal antibody labelled with the same prosthetic group.
CONCLUSIONS: Overall, the small Poly-L-Lysine-based effector molecule (10 kDa) holds the most promise for future research, exhibiting significantly lower uptake in the kidneys and spleen compared to the larger effector (21 kDa) while maintaining an in vivo stability of the astatine-carbon bond comparable to or better than intact antibodies. A proof of concept in vitro cell study demonstrates rapid reaction between the small astatinated effector and a TCO-labelled antibody, indicating the potential of this novel Poly-L-Lysine-based pretargeting system for further investigation in an in vivo tumor model.

Targeted alpha particle therapy (TAT) has emerged as a promising strategy for the treatment of prostate cancer (PCa). Actinium-225 (225Ac), a potent alpha-emitting radionuclide, may be incorporated into targeting vectors, causing robust and in some cases sustained antitumor responses. The development of radiolabeling techniques involving EDTA, DOTA, DOTPA, and Macropa chelators has laid the groundwork for advancements in this field. At the forefront of clinical trials with 225Ac in PCa are PSMA-targeted TAT agents, notably [225Ac]Ac-PSMA-617, [225Ac]Ac-PSMA-I&T and [225Ac]Ac-J591. Ongoing investigations spotlight [225Ac]Ac-hu11B6, [225Ac]Ac-YS5, and [225Ac]Ac-SibuDAB, targeting hK2, CD46, and PSMA, respectively. Despite these efforts, hurdles in 225Ac production, daughter redistribution, and a lack of suitable imaging techniques hinder the development of TAT. To address these challenges and additional advantages, researchers are exploring alpha-emitting isotopes including 227Th, 223Ra, 211At, 213Bi, 212Pb or 149Tb, providing viable alternatives for TAT.