Preclinical and Clinical Status of PSMA-Targeted Alpha Therapy for Metastatic Castration-Resistant Prostate Cancer

Simple Summary Currently, there is no treatment that can cure patients with late stage metastatic prostate cancer. Prostate-specific membrane antigen is a type of protein overexpressed on the membrane surface of most prostate cancer cells. The preclinical and clinical experiences in the rapidly evolving field of targeted alpha-particle radiation therapy for metastatic prostate cancer overexpressing prostate-specific membrane antigen are reviewed. Targeted alpha therapy employs radionuclides emitting highly energetic alpha-particles (cytotoxic payload) chelated to small molecules or monoclonal antibodies designed to target prostate-specific membrane antigen. In this review, we summarize the availability of therapeutic alpha-emitting radionuclides (terbium-149, astatine-211, bismuth-212 (lead-212), bismuth-213, radium-223, actinium-225, thorium-227), and the development of small molecules and antibodies targeting prostate-specific membrane antigen. The limitations in studies using animal models of human prostate cancer to accurately predict efficacy and toxicity in patients are addressed. We have attempted to also critically discuss hurdles related to logistical and supply aspects between different alpha-emitting prostate-specific membrane antigen-targeting radiopharmaceuticals. Lastly, we discuss the potentials, limitations, and future perspectives of prostate-specific membrane antigen-targeted alpha therapy. Abstract Bone, lymph node, and visceral metastases are frequent in castrate-resistant prostate cancer patients. Since such patients have only a few months’ survival benefit from standard therapies, there is an urgent need for new personalized therapies. The prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer and is a molecular target for imaging diagnostics and targeted radionuclide therapy (theragnostics). PSMA-targeted α therapies (PSMA-TAT) may deliver potent and local radiation more selectively to cancer cells than PSMA-targeted β− therapies. In this review, we summarize both the recent preclinical and clinical advances made in the development of PSMA-TAT, as well as the availability of therapeutic α-emitting radionuclides, the development of small molecules and antibodies targeting PSMA. Lastly, we discuss the potentials, limitations, and future perspectives of PSMA-TAT.


Introduction
Prostate cancer is the second most common cancer in men worldwide, with an estimated 1.3 million new cases and 359,000 deaths in 2018 [1]. The tumors of 10-20% of prostate cancer patients become refractory to androgen deprivation therapy and progress as metastatic castration-resistant prostate cancer (mCRPC) [2,3]. Bone metastases dominate, but lymph node and visceral metastases are also frequent in mCRPC patients [4][5][6]. Treatment options for mCRPC have expanded rapidly in the last 20 years [7][8][9][10][11]. The US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have glands, brain, and intestines [50] but is increased in prostate cancer [21,23,32]. PSMA expression is correlated with prostate growth and significantly associated with the degree of differentiation and progression of mCRPC [29]. The precise mechanisms of the physiological function of PSMA in prostate cancer and its regulation are unknown. It has been suggested that elevated levels of PSMA enzymatic activity in prostate cancer cells increases cell folate uptake and proliferation and contributes to prostate carcinogenesis and progression [51]. Figure 1. The simplified structure of prostate-specific membrane antigen (PSMA), its binding sites for PSMA ligands and antibodies (this figure is based on references [52][53][54]).

Targeted Radionuclide Therapy of Prostate Cancer
Targeted radionuclide therapy (TRT) is a rapidly growing treatment modality for mCRPC [33,55,56]. TRT involves the use of radioisotopes or radiolabeled compounds that either naturally accumulate in or are designed to target and deliver a cytotoxic amount of radiation to prostate cancer cells sparing the surrounding normal tissues [55,56]. The systemic administration of tumor-targeted radiopharmaceuticals allows the simultaneous treatment of wide-spread bone and extraskeletal metastases [56,57], whereas a multiple and large-field irradiation using external beam radiotherapy is too toxic [58]. Currently, the β --emitting radionuclide 177 Lu is the most clinically used for TRT [37,38]. However, α particles present significantly higher energies than βparticles, which combined with very short path lengths (<0.1 mm) result in high LET and a greater probability of generating DNA double-strand breaks upon interaction with cell nuclei (Table 1). This occurs almost independently of tissue oxygenation, dose rate, and cellular resistance to photon irradiation and chemotherapy [59]. Beta particle irradiation produces mainly single-strand breaks, exhibiting lower cytotoxic potency than α particles (Table 1). Consequently, the use of α emitters allows the specific targeting and killing of individual

Targeted Radionuclide Therapy of Prostate Cancer
Targeted radionuclide therapy (TRT) is a rapidly growing treatment modality for mCRPC [33,55,56]. TRT involves the use of radioisotopes or radiolabeled compounds that either naturally accumulate in or are designed to target and deliver a cytotoxic amount of radiation to prostate cancer cells sparing the surrounding normal tissues [55,56]. The systemic administration of tumor-targeted radiopharmaceuticals allows the simultaneous treatment of wide-spread bone and extraskeletal metastases [56,57], whereas a multiple and large-field irradiation using external beam radiotherapy is too toxic [58]. Currently, the β − -emitting radionuclide 177 Lu is the most clinically used for TRT [37,38]. However, α particles present significantly higher energies than β − particles, which combined with very short path lengths (<0.1 mm) result in high LET and a greater probability of generating DNA double-strand breaks upon interaction with cell nuclei (Table 1). This occurs almost independently of tissue oxygenation, dose rate, and cellular resistance to photon irradiation and chemotherapy [59]. Beta particle irradiation produces mainly single-strand breaks, exhibiting lower cytotoxic potency than α particles (Table 1). Consequently, the use of α emitters allows the specific targeting and killing of individual malignant cells while minimizing the toxicity to surrounding healthy tissue. Therefore, α particles are highly cytotoxic and promising candidates for TRT.

Radionuclides Used for PSMA-TAT
Ra-223 (Xofigo) is the first-in-class and only α-emitting radiopharmaceutical currently approved by the FDA and the EMA for the treatment of mCRPC patients with symptomatic bone metastases and no known extraskeletal metastatic disease [63][64][65]. Ra-223 has a natural affinity for areas of high bone turnover because it is a bone-seeking calcium mimetic [66]. Targeted 223 Ra therapy for mCRPC with its overall survival benefit has revolutionized the field of TRT [67,68]. Regional lymph node and visceral metastases, most frequently in lung and liver, are seen in approximately 8-15% of newly diagnosed patients but increase over time, currently affecting close to half of men during the course of their disease and represent the lethal phase of the disease [69,70]. Extraskeletal metastatic disease is now of growing concern in patients with mCRPC failing chemotherapy and/or the new oral anti-hormonal therapies targeting the androgen receptor axis (abiraterone and enzalutamide) evident by recent developments in diagnostic nuclear medicine; i.e., PSMA-positron emission tomography (PET) imaging [69,70]. An ideal TAT for mCRPC must exhibit a dual mode of action to combat the entire spectrum of metastases, both micrometastases and overt metastatic disease in lymph nodes, the skeleton, and visceral organs. The majority of bone, visceral, and lymph node metastases highly express PSMA [21,22]. Several α-emitting radionuclides, 149 Tb, 211 At, 212 Pb/ 212 Bi, 223 Ra, 225 Ac, and 227 Th, and PSMA targeting agents are now being actively evaluated for PSMA-TAT (Tables 2-5). The availability, physical and chemical properties of radionuclides play an important role in identifying α-emitting radionuclides suitable for therapy. The half-life of the radionuclide should neither be too long to avoid extended radiation effects after radiopharmaceutical administration nor too short to have enough time for logistical aspects, such as production and transportation. The radiopharmaceuticals must be sufficiently stable compounds both in vitro and in vivo. The nuclear recoil energy of about 100-200 keV from α-decay is sufficient to break chemical bonds between the targeting moiety and the radionuclide, which can lead to circulating carrier-free daughter radionuclides in blood [114,[118][119][120][121]. The decay of the parent radionuclides 212 Pb/ 212 Bi, 225 Ac, 223 Ra, and 227 Th form multiple α-emitting daughter radionuclides ( Figure 2). The released daughter radionuclide can be retained inside the tumor and enhance the cytotoxic effect, but if the released daughter radionuclide has a long enough half-life, it can redistribute within the body and damage healthy tissues [118,120,122]. The radiopharmaceuticals must be sufficiently stable compounds both in vitro and in vivo. The nuclear recoil energy of about 100-200 keV from α-decay is sufficient to break chemical bonds between the targeting moiety and the radionuclide, which can lead to circulating carrier-free daughter radionuclides in blood [114,[118][119][120][121]. The decay of the parent radionuclides 212 Pb/ 212 Bi, 225 Ac, 223 Ra, and 227 Th form multiple α-emitting daughter radionuclides ( Figure 2). The released daughter radionuclide can be retained inside the tumor and enhance the cytotoxic effect, but if the released daughter radionuclide has a long enough half-life, it can redistribute within the body and damage healthy tissues [118,120,122].   149 Tb, 211 At, 225 Ac, 227 Th, 223 Ra, and 224 Ra radionuclides. α-alpha particle, β-beta particle, β − -electron, β + -positron, ε-electron capture. Information extracted from the National Nuclear Data Center database, Brookhaven National Laboratory [75]. Table 3. Comparisons of prostate-specific membrane antigen (PSMA)-targeting small molecules, antibodies, and their fragments [55,[76][77][78][79][80].

PSMA Targeting Agents in TRT
The extracellular domain makes up to 95% of PSMA and provides an accessible target for small molecules, antibodies, and their fragments [29,80]. A number of PSMA-targeting small molecules (also denoted as ligands or inhibitors) and antibodies have been developed for molecular imaging by single-photon emission computed tomography (SPECT) and PET and/or TRT [29,30,123,124]. Small molecules offer potential advantages over antibodies, such as small size, easy synthesis and modification, high specificity and affinity, good permeability with rapid accumulation in tumor, and rapid clearance from most normal tissues (Table 3).
Many different PSMA ligands have been developed [124][125][126][127], and several of them have been investigated for TAT in preclinical and clinical studies (Tables 4 and 5). Small molecule PSMA ligands consist of three components: a binding entity (motif), a linker (spacer), and a radiolabel-bearing moiety (a bifunctional chelator for radiolabeling with metal radionuclides or a prosthetic group for astatination) [41,125,126,128]. The linker region is used to adjust the molecular size and polarity to impact the in vivo distribution properties [125][126][127][129][130][131][132][133]. The first small molecule inhibitors targeting PSMA, based on the glutamate-urea-lysine entity (MIP-1072 and MIP-1095), were introduced into the clinic in 2013 for prostate cancer imaging with 123 I [134]. These inhibitors showed a rapid advantageous localization in tumor lesions, including soft tissue and bone metastases. In 2014, German research groups from Heidelberg and Munich developed PSMA-11 (also called PSMA-HBED-CC) and PSMA I&T ligands, respectively [135,136]. In 2015, the Heidelberg group developed PSMA-617 [130]. In 2020, the FDA approved 68 Ga-PSMA-11 as the first drug for PET imaging of PSMA positive lesions in men with prostate cancer [137]. Unfortunately, the chelator HBED-CC (N,N'-bis [2-hydroxy-5-(carboxyethyl)benzyl] ethylenediamine-N,N'diacetic acid) used in PSMA-11 is not suitable for the stable complexation with therapeutic radionuclides [138]. To date, PSMA-617 and PSMA I&T are the most commonly used theragnostic PSMA radioligands because they can be radiolabeled with diagnostic, e.g., 68 Ga and 44 Sc for PET and 111 In for SPECT, as well as therapeutic radiometals [138][139][140][141][142][143][144]. In an attempt to prolong circulation in the blood and therewith, to increase the dose delivered to tumors and the tumor-to-kidney ratio, PSMA ligands have been structurally modified by adding albumin-binding moieties [123,132,[145][146][147][148][149][150]. Preclinical and clinical studies are required to demonstrate whether PSMA inhibitors with enhanced albumin binding (HTK01169, PSMA-ALB-56, RPS-074, EB-PSMA-617, CTT1403) can increase the efficacy of PSMA-TRT [103,146,148,150,151]. A recently published review provides a comprehensive overview of the current status of selected PSMA inhibitors that have been developed from 1996-2020, emphasizing recent synthetic advances and chemical strategies while highlighting the therapeutic potential and drawbacks of each inhibitor [152].
Several antibodies and their fragments are also being evaluated for TRT in preclinical and clinical studies [97,104,153,154]. The antibodies J591, 107-1A4, PSMA-TTC, and their conjugates recognize extracellular epitopes of PSMA ( Figure 1) and are the most investigated for TAT [81,96,97,104,154]. J591, 107-1A4, and PSMA-TTC were described and characterized in 1997 by Liu et al. [155], in 1998 by Brown et al. [156], and in 2020 by Hammer et al. [104], respectively. The half-life of the radionuclide must be matched with the antibody residence time in the tumor to deliver the maximum irradiation dose [157]. Antibodies have a plasma half-life of about 2-7 days, and several days are needed to reach maximum tumor uptake (Table 3). Radionuclides with too short or too long half-lives may lead to suboptimal efficacy or toxicity.

Radium-223 for PSMA-TAT
Among all α-emitting radionuclides, 223 Ra has the most suitable half-life (t 1/2 = 11.4 days), decay properties, and safety based on extensive clinical use as Xofigo for TAT. Ra-223 decays via a chain of five short-lived daughter radionuclides (t 1/2 from 0.5 s up to 36.1 min) to stable 207 Pb, emitting four α particles and two β − particles (Figure 2). No redistribution of radioactive daughters has yet been reported [66,158]. Consequently, high decay energy (27)(28) is released over a short range [66,158,159]. Ra-223 can be produced in large quantities from 227 Ac (t 1/2 = 21.7 years) generator [66]. The half-life of 223 Ra provides sufficient time for its preparation, distribution, and administration to patients. Unfortunately, due to the bone-seeking characteristics of 223 Ra, its clinical use is limited to prostate cancer patients with osteoblastic metastases [66]. Chelate complex formation is essential to treat extraskeletal cancer metastases. A number of bifunctional chelators, which carry a functional unit for the immobilization of the radiometal and a functional group for the covalent attachment to a biological target molecule, have been tested to chelate 223 Ra [160,161]. Unfortunately, 223 Ra, like other alkaline earth metals, forms very weak complexes [160,161]. To overcome this limitation, several nanomaterials, hydroxyapatites, polyoxopalladates, nanozeolites, barium sulfate, and titanium dioxide nanoparticles have been tested for stable incorporation of radium and linking to the targeting vector [84,[162][163][164][165][166]. So far, only one paper has been published investigating the encapsulation of 223 Ra into functionalized nanozeolites for PSMA-TAT [84]. NaA zeolite is one of the synthetic, non-toxic microporous crystalline aluminosilicate zeolites that can accommodate a wide variety of various molecules, including radionuclides [165,167]. Czerwińska et al. [84] modified NaA nanozeolite with silane-PEG (polyethylene glycol) and functionalized it with anti-PSMA D2B antibody as a carrier of 223 Ra and its daughter radionuclides for PSMA-TAT. The obtained 120 nm 223 RaA-silane-PEG-PSMA D2B bioconjugate was highly stable (<2% of 223 Ra and <6% of the daughter radionuclides were released up to 12 days) [84]. The radioimmunoconjugate bound specifically and internalized into PSMA-expressing C4-2 cells, but not into PSMA-negative DU-145 cells. Treatment of C4-2 cells with 20 kBq/mL of the radioimmunoconjugate resulted in an 80% reduction in metabolic activity [84]. Further preclinical studies in vivo are needed to validate the therapeutic efficacy and toxicity of 223 RaA-silane-PEG-D2B. It is too early to know if this approach will be successful.

Bismuth-213 for PSMA-TAT
Bi-213 is a mixed α and β − emitter with a half-life of 45.6 min (Figure 2). Preclinical studies have demonstrated that TAT with 213 Bi labeled antibody J591, small molecule inhibitor PSMA-I&T or nanobody JVZ-008 showed efficient and rapid tumor targeting, induced apoptosis in PSMA-overexpressing cell lines, significantly delayed spheroid growth of xenograft tumor growth in nude mice [81,[96][97][98]. Small molecule PSMA-I&T induced more double-strand breaks than nanobody JVZ-008 [98]. A dosimetry estimate comparing 213 Bi-PSMA-617 and 225 Ac-PSMA-617 has demonstrated that the short-lived 213 Bi is an inferior choice for TAT with PSMA-617 [168]. To our knowledge, there is only one single case report to date on the clinical application of 213 Bi-PSMA-617 [105]. The patient was treated with two cycles of 213 Bi-PSMA-617 with a cumulative activity of 592 MBq. A biochemical response (decrease in PSA level from 237 µg/L to 43 µg/L) was observed [105]. The short half-life of 213 Bi makes this radionuclide less suitable for routine therapeutic applications due to logistical challenges. Hence, the subject is not discussed further.

Actinium-225 for PSMA-TAT
Ac-225 (t 1/2 = 10 days) decays via a chain of five daughter radionuclides (t 1/2 from 3.7 µs up to 45.6 min) to stable 209 Bi, emitting four α particles and two β − particles ( Figure 2). Earlier, the potential use of 225 Ac for TAT was limited because of difficulties in the selection of chelating agents able to form strong bonds, leading to renal toxicity induced by the longer-lived decay product 213 Bi [55,121]. Nevertheless, some stable complexes have been synthesized, and several more recent clinical trials have demonstrated the potential of 225 Ac-PSMA-617 and 225 Ac-PSMA-I&T for the treatment of mCRPC ( Table  5). The first-in-human PSMA-TAT study was published in 2016 by Kratochwil et al. [42] from Heidelberg. Two mCRPC patients with challenging clinical situations and extensive pretreatment were treated with 100 kBq/kg of 225 Ac-PSMA-617 at bi-monthly intervals as salvage therapy after the presence of a PSMA-positive tumor phenotype had been validated by 68 Ga-PSMA-11 PET/CT [42]. The first patient was not suitable for 177 Lu-PSMA-617 (diffuse red marrow infiltration), and the second one was resistant to 177 Lu-PSMA-617 [42]. Both patients showed a complete response on the PET/CT scan, and PSA declined below the measurable level [42]. Salivary gland toxicity leading to dry mouth syndrome or xerostomia was reported in both patients [42]. The second study with 14 mCRPC patients found that a treatment activity of 100 kBq/kg of body weight of 225 Ac-PSMA-617 per cycle every 8 weeks was the most optimal when considering both efficacy (biochemical response) and tolerability [106]. Severe xerostomia was the dose-limiting toxicity [106]. This standardized treatment protocol for 225 Ac-PSMA-617 is routinely applied for salvage therapy of end-stage mCRPC patients in many studies [106,107,113]. The efficacy of 225 Ac-PSMA-617 TAT was evaluated in 40 mCRPC patients [107]. This study demonstrated a PSA decline of more than 50% in 63% of patients, with a median duration of tumor control of 9 months [107]. The median overall survival was more than 12 months [107]. The majority of mCRPC patients at the Heidelberg clinic were heavily pretreated before TAT with chemotherapy, radiotherapy, and androgen deprivation therapy (abiraterone (85%) and enzalutamide (60%)) [107]. In South Africa, chemotherapy-naïve mCRPC patients treated with 225 Ac-PSMA-617 TAT had reduced toxicity to salivary glands, a 90% PSA decline in 88% of the patients, and 50% achieved undetectable serum PSA and remained in remission 12 months after therapy [109]. The first clinical data using 225 Ac-PSMA-I&T showed highly comparable biochemical responses as after 225 Ac-PSMA-617 TAT [116,117].
Since PSMA-617 crosses the blood-brain barrier and accumulates in cerebral metastases [171], a significant regression of cerebral metastases was demonstrated using 225 Ac-PSMA-617 [108]. Prostate cancer patients with brain metastases have limited treatment options and poor survival, and TAT with 225 Ac-PSMA-617 may have substantial therapeutic potential for these patients.
In the clinical setting, several studies reported toxicity related to TAT with 225 Ac-PSMA-617/PSMA-I&T (Table 5). Xerostomia is a common side effect that causes 10-25% of patients to stop TAT with 225 Ac-PSMA [42,107,111,112,117,172]. Xerostomia should, therefore, be prevented. Modification of the administered activity of 225 Ac-PSMA-617 and the number of cycles of TAT may decrease the side effects while still achieving response [111,173]. Sialendoscopy with dilatation, saline irrigation, and steroid injection (prednisolone) have been investigated in patients with some but limited success [174]. A case report in one patient describes the potential beneficial effects of intraparenchymal injections of botulinum toxin before 225 Ac-PSMA-617 TAT [175]. External cooling of the salivary gland using ice packs from 30 min pre-infusion through 2 h post-infusion of radiopharmaceuticals was expected to reduce PSMA radioligand uptake due to vasoconstriction [172]. However, the relative contributions of salivary gland cooling and the reduced 225 Ac-PSMA-617 activity in minimizing xerostomia severity remain unclear. Therefore, effective methods to reduce salivary toxicity are needed.
Due to the physiological expression of PSMA in kidneys and predominantly renal excretion of 225 Ac-PSMA-617, there is concern about possible radiation toxicity to the kidneys that may cause acute and long-term effects [138,176,177]. It has been reported that the kidney function deteriorated in a patient with one functional kidney after 225 Ac-PSMA-617 [109] and that chronic kidney disease was found in two patients with mCRPC after 225 Ac-PSMA-617 therapy [114]. Until now, retention times of PSMA ligands either in kidneys or in tumor cells have not yet been evaluated systematically [168]. If PSMA on the surface of cancer cells is not sufficiently internalized after binding of the ligand, TAT with 225 Ac with multiple unstable daughters might be suboptimal and toxic [168]. It has also been speculated that the radioactive daughters of 225 Ac, but not 225 Ac-PSMA-617, can accumulate in the tubular cells and irradiate the kidneys, leading to renal injury [114]. In such cases, the therapeutic potential of 225 Ac-PSMA will be substantially decreased, and toxicity increased. Further studies are necessary to evaluate the stability, retention times, and intracellular localization in cancer cells and kidneys of the 225 Ac-PSMA complexes intended for TAT.
The first report on the use of 225 Ac-PSMA-617 in vivo was only published in 2019 [99]. NSG mice bearing subcutaneous C4-2 tumors were treated with 20, 40, and 100 kBq/mouse of 225 Ac-PSMA-617 [99]. Significant tumor growth inhibition was observed in all treatment groups compared to the control. However, mice treated with 100 kBq had some weight loss, while the mice treated with lower activities experienced only transient weight loss. In 2020, the same group reported a useful mouse model of human metastatic prostate cancer by injecting C4-2 cells expressing firefly luciferase into the left ventricle of NSG mice, which was then used to evaluate the effectiveness of 225 Ac-PSMA-617 at various disease stages [100]. Early treatment, one-week post-inoculation of C4-2 cells, with 40 kBq/mouse of 225 Ac-PSMA-617 prevented liver metastases and led to significant survival benefit [100]. In 2019, Kelly et al. [103] studied the albumin-binding and PSMA-targeting ligand RPS-074 labeled with 225 Ac in BALB/c mice bearing LNCaP xenograft tumors. Significant tumor growth inhibition was observed in mice treated with 74 and 148 kBq of 225 Ac-RPS-074. A single injection of 148 kBq induced a complete response in 6 of 7 tumors, with no apparent toxic effects. In 2020, Current et al. [101] documented that 225 Ac-PSMA-617 efficacy is dependent on cellular PSMA levels and intra-tumoral PSMA heterogeneity.
Another approach to reducing toxicity in salivary glands and kidneys is to use antibodies (e.g., J591) instead of small molecule PSMA for TAT [83,154]. J591 binds to a different site of PSMA compared to PSMA ligands and has a much lower distribution in salivary glands and kidneys [154]. Ongoing clinical trials with 227 Ac-J591 will provide the missing information on stability, efficacy, and toxicity (Table 6). The mainly retrospective studies have reported promising response rates, progressionfree survival, and overall survival ( Table 5). The limited availability of 225 Ac is the main challenge for its clinical use. Many research centers investigate the possibility of producing 225 Ac in commercial quantities. In the future, scaled-up production of 225 Ac could be achieved by the use of a high current cyclotron or electron linear accelerator (linac) [178,179].

Thorium-227 for PSMA-TAT
Th-227 has a physical half-life of 18.7 days and decays through radioactive 223 Ra and the other short-lived radionuclides in its decay chain to stable 207 Pb by emitting five α particles ( Figure 2). The long half-life of 227 Th allows transportation and preparation of the radiopharmaceutical. Due to its chemical properties, 227 Th can be linked to a variety of antibodies and proteins [72,180]. Th-227 complexed with octadentate 3,2-hydroxypyridinone (3,2-HOPO) chelators that are conjugated to antibodies or other targeting moieties results in highly stable targeted 227 Th conjugates (TTCs) [104]. TTCs, therefore, represent a new promising class of TAT [104]. A novel, fully human antibody-based TAT, PSMA-TTC (BAY 2315497), has been recently developed by Bayer and tested in mice bearing prostate cancer tumors [104]. The antitumor efficacy of PSMA-TTC was observed in different prostate cancer models. However, survival and long-term toxicity have not been reported. These preclinical data encouraged the further investigation of BAY 2315497 in an ongoing phase I trial in mCRPC (Table 6).

Terbium-149 for PSMA-TAT
A very interesting alternative to the presented α emitters is the 149 Tb, currently studied in preclinical radioimmunotherapy [85]. Tb-149 has a half-life of 4.1 hours and is considered a promising theragnostic radionuclide. It decays by α emission (3.97 MeV, 16.7%), as well as electron capture (76.2%), positron emission (7.1%), gamma rays, and X-rays. Therefore, it can be used for TAT and PET imaging [181]. Longer half-life of 149 Tb compared to 213 Bi (t 1/2 = 46 min) and 212 Bi (t 1/2 = 1.0 h), absence of α-emitting daughters, and chelation with S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) are clear advantages of 149 Tb [181]. Antitumor activity of 149 Tb-PSMA-617 has been demonstrated in mice bearing PC3 PIP tumors [85]. Tb-149 is a rare-earth element, and its production and chemical separation are associated with serious difficulties, which partially explains why 149 Tb is not in clinical use yet [182]. Additionally, the presence of long-lived daughters appearing during the decay of 149 Tb (Figure 2) complicates dosimetry and might increase the risk of undesirable physiological effects. The long-lived daughter radionuclides, 149 Eu (t 1/2 = 93 d) and 145 Sm (t 1/2 = 340 d), are potential bone seekers [183]. Due to the recoil energy of the alpha decay of 149 Tb, there is a high risk of release of these radionuclides into the bloodstream leading to potential accumulation in bone and, thus, to long-term irradiation of the bone marrow [183].

Lead-212 for PSMA-TAT
Another suitable radionuclide for PSMA-TAT is β − emitter 212 Pb (t 1/2 = 10.6 h) that acts as an in vivo generator of α particles via its short-lived progenies 212 Bi (t 1/2 = 60.6 min) and 212 Po (t 1/2 = 0.3 µs) (Figure 2). The decay of one 212 Pb atom releases on average one α particle and a mean α energy of 7.9 MeV. The use of 212 Pb circumvents the impractical short half-life of 212 Bi and delivers around ten times the dose per administered activity unit compared to 212 Bi alone, reducing the amount of activity required [184,185]. Pb-212 can be obtained at an industrial scale from 224 Ra-based generators using 228 Th (t 1/2 = 1.9 years) as a long-term generator. It has suitable properties in terms of chelation chemistry and forms stable complexes both with the versatile chelator DOTA and the lead-specific chelator S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra(2-carbamoylmethyl)cyclododecane (TCMC) [186]. Comprehensive reviews describing the use of 212 Pb for TAT of cancer have recently been published [170,187,188].
Several research groups have developed PSMA-targeting ligands specifically designed for 212 Pb chelation that show promising tumor-targeting in preclinical models [91,92,94,95]. The urea-based PSMA ligands NG001, labeled with 212 Pb, and CA012 and L2, labeled with the surrogate 203 Pb, show high tumor uptake (8-25%ID/g) 1-2 h post-injection in mice bearing PSMA-positive tumors, which is comparable with clinically used 177 Lu-PSMA-617 [91,92,94,130,148]. In NSG mice bearing PC3 PIP tumors, 1.5 and 3.7 MBq of 212 Pb-L2 significantly inhibited tumor growth with therapeutic indices (TI) of 1.9 and 3.0, respectively, with time to reach a 10-fold tumor increase used as an endpoint [92]. In a micrometastatic tumor model, 3.7 MBq of 212 Pb-L2 demonstrated an increased survival benefit compared to 37 MBq of 177 Lu-PSMA-617 (TI of 1.2 vs. 1.0, respectively). However, a long-term toxicity study of 212 Pb-L2 in healthy, immunocompetent mice identified the kidney as the dose-limiting organ, and the maximum tolerated dose (MTD) of the radioligand was determined to be 1.5 MBq. In athymic nude mice bearing C4-2 xenografts, an injected activity of only 0.32 MBq of 212 Pb-NG001 demonstrated increased survival compared to the control group with a therapeutic index of >2.0 (median survival of 15 days vs. >30 days) [93]. The results warrant further preclinical studies to evaluate the long-term toxicity of 212 Pb-NG001.
With shorter-lived radionuclides, such as 212 Pb, the high initial kidney uptake could present a potential toxicity problem because of the higher dose rate of these radionuclides [189]. However, the mentioned Pb-labeled ligand NG001 exhibit lower kidney uptake than PSMA-617 [94]. Promising therapeutic results labeled with the short-lived 213 Bi (t 1/2 = 45.6 min) with an even higher dose rate than 212 Pb were reported [105]. Another challenge with 212 Pb is the retention of daughter radionuclides in the chelator after decay. Up to 36% of 212 Bi could dissociate from DOTA and TCMC chelators of antibody complexes because of high recoil energies of the α-emitting daughters [120,184,190,191]. However, no translocation of the 212 Bi daughter was detected in non-targeted organs during a 24 hour study period of 212 Pb-NG001, likely prevented by the rapid tumor targeting and cellular internalization, as well as the rapid normal tissue clearance of the radioligand [94].

Dual Alpha ( 224 Ra& 212 Pb) for TAT
An ideal PSMA-TAT for mCRPC must combat the entire spectrum of metastases present in the patients. A dual-alpha approach that uses the high LET ionizing energy of 224 Ra and daughter radionuclides to target various mCRPC lesions have been presented [95]. Here, a 224 Ra solution in transient equilibrium with daughter radionuclides was used for in situ labeling of a PSMA-targeting ligand, i.e., 212 Pb is complexed by the ligand in the presence of 224 Ra [95,184,185]. The resulting solution has dual-targeting properties; natural bone-seeking 224 Ra will target osteoblastic metastatic lesions, and the 212 Pb-labeled PSMA ligand will target extraskeletal metastases by selective binding to the surface of PSMA-expressing cells [95]. Thus, the dual-alpha approach is providing a boneseeker and a PSMA-seeker in one radiopharmaceutical solution. The accumulation of 224 Ra in bone and 212 Pb-labeled PSMA ligand in tumor sites was verified in C4-2 tumor-bearing mice and warrants further investigation in vivo [95].

Combination Treatments with PSMA-TAT
Around 20% of patients treated with 225 Ac-PSMA-617 have a poor response (Table 5) or early resistance against 225 Ac-PSMA-617 [107], despite sufficient expression of PSMA and uptake of 225 Ac-PSMA-617 in their tumors [192]. Several combination treatments with PSMA-TAT have been proposed [173,192,193]. Their goal is to increase PSMA-TAT efficacy by using therapies with different action mechanisms together with TAT, keeping toxic effects to a minimum. The tandem PSMA-RLT approach has been introduced to increase efficacy and reduce toxicity [173,194]. A single course of tandem therapy with low-activity 225 Ac-PSMA-617 and full-activity 177 Lu-PSMA-617 has enhanced response to PSMA-RLT and minimized xerostomia severity in men with late-stage/end-stage mCRPC [173,194]. There is a subgroup of patients (~17%) with poor response to 225 Ac-PSMA-617 who harbor mutations in DNA damage-repair and checkpoint genes [192]. Combining PSMA-TAT and DNA damage-repair-targeting agents, such as poly(ADP-ribose)-polymerase inhibitors, have been suggested for these patients [192]. Lastly, it has been hypothesized that PSMA-TAT may increase tumor immunogenicity, and the use of immune checkpoint inhibitors may improve efficacy [102]. Synergistic antitumor efficacy between 225 Ac-PSMA-617 and PD-1 blockage has recently been observed in C57BL/6-mice bearing syngeneic RM1-PGLS tumors [102].

Limitations of Preclinical Studies Related to Clinical PSMA-TAT
A major concern of PSMA-targeting radioligands is the radiotoxicity in PSMA-expressing organs. Only the most optimal and safest PSMA-targeted radioligands from preclinical studies are tested in humans. However, radiotoxicity in rodents is not the most accurate predictor of toxicity in humans because the human, rat, and mouse PSMA have different patterns of anatomical expression in normal tissues [195][196][197]. For example, PSMA levels in human submandibular gland are approximately four-fold higher compared with mouse and fifty-four-fold higher compared with rat submandibular gland [197]. Rupp et al. demonstrated that the accumulation of PSMA ligands in salivary glands in humans is high due to PSMA-unrelated uptake mechanisms [198], while PSMA radioligands ( 177 Lu, 212 Pb) show negligible uptake in mouse salivary glands [94,95,132]. The details of salivary glands' uptake mechanisms are currently unclear and must be further investigated using different clinical models. Furthermore, PSMA levels in human kidneys are approximately two-fold lower compared with mouse kidneys [138]. Many patients treated with PSMA-TAT report severe xerostomia, and only a few of them, nephrotoxicity ( Table 5). The direct comparison of PSMA-TAT data from various groups is not easy since different mouse strains, cell lines, and different specific activities of radiopharmaceuticals have been used in the various studies. PSMA-TAT efficacy increases with increasing PSMA levels [101]. For example, transduced PC3 PIP cells present much higher PSMA expression levels than LNCaP or C4-2 cells (Table 7), and the direct comparisons of survival, tumor uptake, and tumor to kidney ratio of different radiopharmaceuticals must be done carefully. Table 7. PSMA expression in prostate cancer cell lines. N/A, not applicable; negative, below the lower limit of quantification.

Future of PSMA-TAT
The success of PSMA-TAT depends on the availability of α emitters and chelators, enhanced tumor uptake of linkers and targeting moieties, and reduced toxicity and progeny redistribution.
Further preclinical proof of concepts with PSMA-TAT in relevant murine models of mCRPC regarding the impact of dose rate and relative biological effectiveness is sorely needed. This also relates to direct comparisons of 225 Ac, 227 Th, and 212 Pb PSMA-TAT with 177 Lu-PSMA TRT.
The detailed mechanisms of action of PSMA-TAT are far from fully understood [72]. Elucidating in vitro and in vivo molecular and cellular mechanisms related to targeted and bystander effects of PSMA-TAT are important. It has been suggested that TAT bears the potential to induce immunogenic cell death through the release of damage-associated molecular patterns from dying tumor cells that result in the activation of tumor-specific immune responses [207]. However, the role of PSMA-TAT in modulating the immunogenicity of prostate cancer cells remains unknown.
The dosimetry for the labeled α emitter and its progeny is challenging and still in the early stage because of the heterogeneous antigen expression among cancer cells and the nature of short-range, high-LET alpha radiation (Tables 1 and 2). This needs to be further investigated using modeling methods. Autoradiographs can help to obtain high-resolution images, but they can only be performed ex vivo. In such cases, estimates of radiation doses could be based on experimental data and modeling. Recommended therapeutic activities (e.g., 4-8 MBq 225 Ac-PSMA-617) limit the clinical applicability of SPECT [208]. Theranostic imaging protocols using 68 Ga-PSMA ligands does not provide any information about the translocation of daughter radionuclides, and the impact of recoiling daughter molecules on dosimetry of α-emitting radiopharmaceuticals on healthy tissues are unknown.

Conclusions
Preclinical studies and preliminary efficacy and safety data on PSMA-TAT in mCRPC patients are very encouraging. Because of production cost, logistical, and supply problems with several of the more promising α-emitters, the future of clinical PSMA-TAT should focus on radionuclides that are suitable for large scale production and supply, such as 212 Pb, which can be produced at the industrial scale with existing methods. Dual targeting with 212 Pb/ 224 Ra is particularly promising since most late-stage prostate cancers have skeletal involvement, and 212 Pb-PSMA ligand and 224 Ra represent two different targeting approaches in one radiopharmaceutical solution. The future of clinical PSMA-TAT may also include novel PSMA ligands and combined approaches.