Preclinical Efficacy of a PSMA-Targeted Thorium-227 Conjugate (PSMA-TTC), a Targeted Alpha Therapy for Prostate Cancer

Despite recent progress, a high medical need remains for novel, efficient prostate cancer therapies particularly in advanced disease. One of the well-established target antigens in prostate cancer is prostate-specific membrane antigen (PSMA; FOLH1), a type II transmembrane glycoprotein that acts as a glutamate carboxypeptidase. It is highly and specifically overexpressed on the cell membrane of prostate cancer cells, including castration-resistant and metastatic stages of the disease (1). Recently, PSMA-targeted radiotherapy using both antibody and peptide-based approaches has attracted increasing interest (1). In particular, the small-molecule PSMA-617 radiolabeled with the beta emitter lutetium-177 (¹⁷⁷Lu-PSMA-617) has demonstrated promising prostate-specific antigen (PSA) responses (2) and is now undergoing pivotal trials (NCT03511664) in metastatic castration-resistant prostate cancer (mCRPC).

However, there have been clinical signs that alpha emitters can surpass beta radiation in efficacy (3, 4), as the alpha emitter actinium-225–labeled PSMA-617 (²²⁵Ac-PSMA-617) has shown activity in individual patients refractory to beta-emitting ¹⁷⁷Lu-PSMA-617 therapy, and a high response rate has been observed in initial ²²⁵Ac-PSMA-617 studies (3, 4). This might be explained by the higher linear energy transfer of alpha emitters and their ability to induce clustered DNA double-strand breaks (DSB), which are particularly difficult for cells to repair, in contrast to beta emitters which induce mainly single-strand DNA breaks. Additionally, the proven survival benefit and approval of radium-223 dichloride (Xofigo) for treatment of mCRPC in patients with symptomatic bone metastases and no known visceral metastases support the great potential of targeted alpha therapy (TAT) for the treatment of prostate cancer (5).

In the clinic, the application of ²²⁵Ac-PSMA-617 is unfortunately limited by severe xerostomia, which, due to strong uptake of the PSMA-targeting ligands into the salivary glands, becomes a dose-limiting toxicity (3, 6). In contrast, with PSMA-targeting antibody formats, the observed salivary gland uptake is very low (6–9). Therefore, TAT with a monoclonal antibody has the potential to complement the portfolio of PSMA-targeting radiotherapeutics and may overcome the xerostomia that limits the use of the radiolabeled small-molecule ligands (6).

Here, we report the preclinical characterization of a novel, antibody-based TAT for the treatment of prostate cancer, namely, the PSMA-targeted thorium-227 conjugate (PSMA-TTC) BAY 2315497. PSMA-TTC uses the alpha-particle emitter thorium-227 (half-life of 18.7 days), which is the parent nuclide of radium-223. Thorium-227 can be efficiently complexed by the 3,2-hydroxypyridinone (3,2-HOPO) octadentate chelator (10, 11) covalently linked to antibodies (12–15) at ambient temperature. Strong antitumor efficacy of PSMA-TTC is shown in a panel of prostate cancer models mimicking different clinical stages of prostate cancer including castration-resistant and bone metastatic disease. Additionally, potential biomarkers, tolerability as well as various doses and schedules, are investigated. Taken together, the data support the clinical development of PSMA-TTC in the treatment of prostate cancer.

The fully human PSMA-targeting antibody BAY 2315158 specifically recognizing human and cynomolgus PSMA was licensed from Progenics Pharmaceuticals, Inc. (16, 17). The nonradiolabeled PSMA antibody–chelator conjugate (BAY 2315493) was manufactured at Bayer AG and Bayer AS by coupling an N-hydroxysuccinimide–activated 3,2-HOPO chelator covalently to the ϵ-amino groups of the lysine residues of the PSMA antibody as described previously (12–15). The radionuclide thorium-227 was manufactured at IFE and the antibody–chelator conjugate was radiolabeled with thorium-227 in citrate buffer (pH 5.5) at +22°C for up to 60 minutes resulting in PSMA-TTC (BAY 2315497) as described in Supplementary Methods and previously (14). A nonbinding radiolabeled isotype control was prepared similarly to PSMA-TTC (12, 14, 15).

The 22Rv1, C4-2, VCaP, and MDA-PCa-2b prostate cancer cells were obtained from ATCC and LNCaP and PC3 cells from DSMZ in 2015 to 2018, and authenticated using short tandem repeat DNA fingerprinting at DSMZ. LNCaP-luc cells were generated by transfecting LNCaP cells with the luciferase gene at NMI. The patient-derived xenograft (PDX) prostate cancer model ST1273 (18) was obtained from South Texas Accelerated Research Therapeutics, KUCaP-1 (19) from O. Ogawa (University of Kyoto, Kyoto, Japan), and LuCaP 86.2 (20) from the University of Washington (Seattle, WA).

Matched samples of primary tumors and lymph node metastases from prostate cancer patients were obtained from Provitro AG.

PSMA-TTC was analyzed for radiostability by instant thin layer chromatography and HPLC as described previously (12). The immunoreactive fraction was determined as previously described (12). The binding affinity of PSMA-TTC to recombinant human PSMA (R&D Systems) was determined with an enzyme-linked immunosorbent assay (ELISA).

In vitro cytotoxicity and caspase experiments were performed using CellTiter-Glo or Caspase-3/7 Glo assay (Promega), respectively, in prostate cancer cell lines after a 5-day exposure to PSMA-TTC. Determination of antibodies bound per cell, cell-cycle analysis (staining C4-2 prostate cancer cells with PI RNAse), and DNA DSBs (percentage of phosphorylated histone protein γ-H2AX–positive LNCaP cells) were performed by flow cytometry as previously described (12, 15). Internalized radioactivity after incubation of C4-2 and PC3 cells with PSMA-TTC was determined as described in Supplementary Methods.

Animal experiments were performed under the national animal welfare laws in Norway, Germany, Finland, and Denmark and approved by the local authorities.

The in vivo biodistribution and antitumor efficacy of PSMA-TTC were evaluated in four human prostate cancer cell line–derived (LNCaP, MDA-PCa-2b, 22Rv1, and C4-2) and three patient-derived (ST1273, KUCaP-1, and LuCaP 86.2) subcutaneous xenograft models with varying PSMA expression levels, and in an intratibial PSMA-expressing prostate cancer xenograft model (LNCaP-luc). For efficacy studies, n = 10 mice/group and for each time point in pharmacokinetic studies n = 3 mice/group were used unless mentioned otherwise.

For the human prostate cancer cell line–derived studies, male CB17-Scid mice (Janvier Labs) were inoculated subcutaneously with 5 × 10⁶ LNCaP, male athymic nude mice (Envigo) with 5 × 10⁶ MDA-PCa-2b, male NMRI Nude mice (Taconic) with 1 × 10⁶ C4-2, and male athymic nude mice (Envigo) or male NMRI nude mice (Janvier Labs) with 3 × 10⁶ or 2 × 10⁶ 22Rv1 cells, respectively.

For the human prostate cancer PDX studies, female NMRI nude mice (Janvier Labs) were implanted subcutaneously with 5 × 5 × 5 mm ST1273, male CB17-Scid mice (Janvier Labs) with 5 × 5 × 5 mm KUCaP-1 or 5 × 5 × 5 mm LuCaP 86.2 tumor fragments.

For the intratibial model, LNCaP-luc human prostate cancer cells (2 × 10⁶) were inoculated into the intratibial bone marrow cavity of male NOD.scid mice (Envigo; n = 10–11 mice/group).

The mice were treated with a single or multiple intravenous (i.v.) injections of PSMA-TTC at 75 to 500 kBq/kg. Unless mentioned otherwise, the TTC and radiolabeled isotype control were administered at a fixed total antibody dose of 0.14 to 0.43 mg/kg. Additionally, the effects of total antibody doses varying between 0.14 and 5 mg/kg were examined.

To circumvent unspecific uptake of the test compound by organs such as spleen, mice were predosed i.v. with 200 μg of an irrelevant mouse antibody (IgG2a-κ, murine myeloma monoclonal UPC10 antibody, Sigma-Aldrich) 16 to 24 hours prior to treatment with TTCs (21).

Subcutaneous tumor growth was monitored by measuring tumor volume (0.5 × length × width²) using a caliper or bioluminescence imaging (BLI) as described in Supplementary Methods. The development of tumor-induced osteoblastic, osteolytic, and mixed lesions in bone was determined by X-ray imaging and microcomputed tomography (micro-CT) in the LNCaP-luc model as detailed in Supplementary Methods. Animal body weight was monitored as an indicator of treatment-related toxicity. Measurement of tumor volume and body weight was performed two to three times per week. Individual animals were sacrificed when showing >20% body weight loss or when tumors reached a maximum size of ∼1,000 mm³. At study termination, the animals were sacrificed by cervical dislocation under CO₂ anesthesia or equal. T/C (treatment/control) ratios were calculated using final tumor volume or tumor burden as determined by bioluminescence analysis.

The levels of PSA were determined in serum samples using the Quantikine Human Kallikrein 3/PSA ELISA kit (R&D Systems) and VICTOR2 Multilabel Counter (PerkinElmer).

Biodistribution and pharmacokinetic studies were performed by measuring remaining thorium-227 radioactivity in tumor, blood, femur, spleen, kidney, liver, and muscle at time points 0.5 hours to 21 days after dosing, were analyzed for remaining radioactivity with high purity germanium gamma detector (HPG) and expressed as a percentage of injected dose of thorium-227 per gram (% ID/g) as described previously (12).

Details of in vivo studies are described in Supplementary Methods.

IHC was performed on paraffin sections of xenograft tumor tissues with anti-human PSMA antibody (clone 3E6, 1:1,000, M3620, Dako Denmark) as described in Supplementary Methods. γ-H2AX and cleaved caspase-3 analyses were performed as previously described in ST1273 tumors (12, 15).

Statistical analysis was performed using R (https://www.R-project.org; R Foundation for Statistical Computing, Vienna, Austria). Data were analyzed using one-way ANOVA followed by either Dunnett test (LNCaP) or ANOVA contrasts (22Rv1); Kruskal–Wallis test followed by Dunn test (MDA-PCa-2b, C4-2); linear models followed by Sidak method (KUCaP-1, LuCaP 86.2, ST1273); or mixed model over all time points followed by model contrasts (LNCaP-luc).

Radiolabeled PSMA-TTC (Supplementary Fig. S1A), nonradiolabeled antibody–chelator conjugate and the nonconjugated PSMA antibody showed equal binding to human PSMA in ELISA experiments (EC₅₀ values approximately 1.2 nmol/L; Fig. 1A). Radiostability analysis showed that PSMA-TTC was stable over 48 hours with expected ingrowth of radium-223, which is released from the chelator upon decay of thorium-227 (Supplementary Fig. S1B). Immunoreactive fraction of the radiolabeled compound was approximately 80%, confirming stable binding to the PSMA target over a 48-hour period (Supplementary Fig. S1C).

The in vitro mode of action of PSMA-TTC was tested in prostate cancer cell lines. Rapid increase in intracellular radioactivity for thorium-227 was observed after incubation of PSMA high-expressing C4-2 cells with PSMA-TTC, but not in PSMA-negative PC3 cells, indicating fast internalization after binding to the receptor (Fig. 1B). A 5-day incubation of prostate cancer cells with PSMA-TTC resulted in an increased proportion of cells staining positive for γ-H2AX, indicating the induction of DNA DSBs (Fig. 1C). Moreover, cell-cycle arrest in the G₂–M phase (Fig. 1D) as well as induction of caspase-3/7 (Fig. 1E) were observed in the PSMA-TTC–treated prostate cancer cells. Exposure to PSMA-TTC reduced the viability of PSMA high–expressing prostate cancer cells (>77,000 antibodies bound per cell) with IC₅₀ values of 0.01 to 0.14 kBq/mL and 15- to 26-fold selectivity compared with radiolabeled isotype control (Supplementary Table S1; Fig. 1F). The selective inhibition of prostate cancer cell viability by PSMA-TTC was dependent on the surface expression of PSMA, as selectivity compared with the radiolabeled isotype control decreased with lower PSMA expression levels. Five-fold selectivity was observed in VCaP cells with moderate PSMA expression (21,000 antibodies bound per cell), 2-fold selectivity in 22Rv1 cells with 2,000 antibodies bound per cell and no selectivity was detected in the PSMA-negative PC3 cells (Supplementary Table S1).

Antitumor efficacy of PSMA-TTC was evaluated in a panel of PSMA-expressing prostate cancer xenograft models representing different disease stages of prostate cancer (Fig. 2A–G; Supplementary Fig. S2A and S2B; Table 1). In the androgen-responsive LNCaP model (high PSMA expression with a H-score of 300), a single injection of PSMA-TTC (at 75, 150, or 300 kBq/kg and total antibody dose of 0.43 mg/kg) resulted in dose-dependent antitumor efficacy (T/C ratios 0.51, 0.42, and 0.31, respectively) with 7 of 10 animals showing partial response or stable disease in the 300 kBq/kg group (Fig. 2A; Table 1). Antitumor activity was selective as the radiolabeled isotype control showed only minor tumor growth inhibition in this model after a single injection (300 kBq/kg; 0.43 mg/kg) with a T/C ratio of 0.8.

To evaluate the antitumor activity of PSMA-TTC in an androgen-independent and castration-resistant setting, its efficacy was determined in the MDA-PCa-2b model (high PSMA expression with a H-score of 300) derived from an androgen-independent prostate cancer bone metastasis, in the castration-resistant 22Rv1 model (moderate/heterogeneous PSMA expression with a H-score of 100–200), and in the castration-resistant variant of LNCaP, namely, the C4-2 model (high PSMA expression, H-score 300). Again, strong antitumor efficacy was observed after a single injection of PSMA-TTC (100, 250, and 500 kBq/kg; 0.14 mg/kg) at T/C ratios of 0.33, 0.18, and 0.09, respectively, for MDA-PCa-2b; and 0.54, 0.37, and 0.19, respectively, for 22Rv1; and 0.27, 0.47, and 0.15, respectively, for C4-2 (Fig. 2B and C; Supplementary Fig. S2A; Table 1). Statistically significant inhibition of tumor growth was seen at doses equaling or above 250 kBq/kg in the MDA-PCa-2b and 22Rv1 models and starting from 100 kBq/kg in the C4-2 model.

To evaluate antitumor efficacy in models mimicking more closely the clinical situation, the efficacy of PSMA-TTC was tested in prostate cancer PDX models. Interestingly, high response rates were observed after treatment with PSMA-TTC in these models. In the androgen-sensitive ST1273 model, marked antitumor efficacy was achieved at a single injection of PSMA-TTC at 125 kBq/kg (T/C 0.3). After a single injection of PSMA-TTC at 500 kBq/kg, a T/C of 0.01 was reached with partial responses observed in 5 of 10 and complete responses in 4 of 10 mice (Fig. 2E). The mean serum PSA level increased in the vehicle and radiolabeled isotype-treated groups in ST1273 tumor–bearing mice during the study, whereas PSMA-TTC treatment at 250 and 500 kBq/kg effectively reduced the serum PSA level (Fig. 2F). Thus, the observed changes in the serum PSA level correlated with the treatment effects on the tumor volume in the same study.

In the enzalutamide-resistant KUCaP-1 model, clear antitumor efficacy was achieved at a single injection of PSMA-TTC at 75 kBq/kg (T/C 0.39). A single injection of PSMA-TTC at 300 kBq/kg resulted in a T/C ratio of 0.07, with 2 of 10 mice showing stable disease and 6 of 10 partial responses (Fig. 2G). Similarly, in the castration-resistant LuCaP 86.2 model, 5 of 10 mice showed stable disease, 1 of 10 partial response, and 1 of 10 complete response (T/C 0.27) after a single injection of PSMA-TTC at 300 kBq/kg (Supplementary Fig. S2B).

The cell line– and patient-derived prostate cancer models harbored PSMA expression with an IHC score of at least 2+ (H-Score >100) as determined by IHC analysis of tumors from vehicle-treated animals (Supplementary Fig. S3A; Table 1). Similar levels of PSMA expression were also observed in more than 85% of tumors or matched lymph node metastases from prostate cancer patients (Supplementary Fig. S3B). This indicates that the PSMA expression levels in the tested models reflect clinically relevant target levels.

Prostate cancer most commonly metastasizes to bone, with up to 90% of mCRPC patients demonstrating bone metastases (22–24). To evaluate the activity of PSMA-TTC in a model mimicking the clinical situation of prostate cancer metastasized to bone, a bone growth model was established by injecting luciferase-labeled LNCaP cells into the mouse tibiae (25). Potent antitumor activity of PSMA-TTC was observed in this model: mice treated with a single dose of PSMA-TTC (100 or 200 kBq/kg) had less LNCaP-luc tumor burden in bone compared with vehicle-treated animals as determined by BLI (Fig. 3A and B). Correspondingly, serum PSA levels were lower in PSMA-TTC–treated as compared with the vehicle-treated mice (Fig. 3C). In the tibiae of vehicle-treated mice, LNCaP-luc tumor growth induced abnormal osteoblastic bone growth as shown by micro-CT analysis and prominently osteoblastic, but also mixed changes representing both osteolytic and osteoblastic components were observed by radiography (Fig. 3D and E). The total area of tumor-induced changes in bone increased steadily in the vehicle group over time, whereas it was remarkably inhibited by both doses of PSMA-TTC (Fig. 3F). A summary of all prostate cancer models used for the assessment of the antitumor efficacy of PSMA-TTC is presented in Table 1.

In the MDA-PCa-2b xenograft model, thorium-227 accumulated in tumors after a single injection of PSMA-TTC (500 kBq/kg, 0.14 mg/kg) with values over 20% ID/g reached after 3 days, increasing to 37% ID/g after 7 days and tumor retention for >21 days (Fig. 4A). In parallel, thorium-227 activity decreased down to 1% ID/g over 7 days in blood. Tumor/organ ratio was above 2-fold for liver and spleen, and 5- to 9-fold for femurs on day 3 after injection and onward. In contrast to this, the tumor uptake observed with the radiolabeled isotype control was below 8% ID/g during the 7 days after injection and the tumor/organ ratio was ∼1 for liver, spleen, and femurs at 7 days after injection (Fig. 4B). Even higher tumor uptake and a tumor/organ ratio were achieved in the ST1273 PDX model with >100% ID/g measured in tumors starting from day 7 after injection onward. These high values were due to shrinking tumor size in PSMA-TTC–treated mice (Supplementary Fig. S4A).

The antitumor efficacy observed in vivo in different prostate cancer models was in line with target-specific tumor uptake and accumulation of PSMA-TTC as observed in the biodistribution studies.

The ratio between thorium-227-labeled and nonradiolabeled PSMA antibody–chelator conjugate can be altered by varying the amount of antibody for a given fixed radioactive dose, leading to potential effects on receptor saturation and pharmacokinetics. Thus, the influence of total antibody doses on biodistribution and antitumor efficacy was tested in the 22Rv1 model with moderate/heterogeneous PSMA expression.

Tumor uptake and retention of radioactivity were found to be comparable (>20% ID/g) at total antibody doses of 0.14 and 0.75 mg/kg between 3 and 21 days after injection of PSMA-TTC at 500 kBq/kg in the 22Rv1 model (Fig. 4C). However, at 5 mg/kg of antibody, there was a significant reduction in tumor delivery with only 6% ID/g retention detected after 21 days (Fig. 4C).

In line with the biodistribution data, a single injection of PSMA-TTC at 500 kBq/kg induced strong tumor growth inhibition in the 22Rv1 xenograft model when administered at a total antibody dose ranging from 0.14 to 1.5 mg/kg (T/C ratios of 0.13–0.31), but showed reduced efficacy when administered at a total antibody dose of 5 mg/kg (T/C 0.84; Fig. 4D; Supplementary Fig. S4B).

In addition, the impact of applying PSMA-TTC at different dosing schedules was investigated. In the ST1273 model, dosing at a single injection of 500 kBq/kq was compared with schedules where the dose was fractionated into 4 weekly (125 kBq/kg) or 2 biweekly (250 kBq/kg) injections. Different dosing schedules resulted in similar effects on tumor growth inhibition in this model (Fig. 4E; Table 1). This observation was confirmed in the LNCaP and KUCaP-1 models (Supplementary Fig. S4C–S4D; Table 1).

Importantly, no significant changes in body weight were observed in the PSMA-TTC–treated groups compared with the vehicle groups, indicating that the applied doses were overall well tolerated in mice (Fig. 2D; Supplementary Fig. S5A). The administration of PSMA-TTC or radiolabeled isotype control resulted in dose-dependent suppression of white blood cells including lymphocytes and neutrophils and reduction of platelets as shown in the ST1273 model (Fig. 4F; Supplementary Fig. S5B–S5E). All hematology values showed a trend toward recovery during the study period with full recovery achieved in the 125 kBq/kg dosed group 57 days after dosing. No effect of treatment on red blood cells was observed during the study duration and the tested dose schedules tested had no impact on the overall effects on hematology values.

The mode of action of PSMA-TTC was confirmed in the ST1273 model in vivo by staining for γ-H2AX and cleaved caspase-3. The induction of DNA DSBs and apoptosis was detected in tumors 7, 14, and 21 days after treatment with PSMA-TTC, whereas only low levels of γ-H2AX and caspase-3 were detected in tumors from mice treated with radiolabeled isotype control or vehicle (Fig. 5A and B).

Recently, there has been growing interest in agents targeting PSMA, a membrane protein expressed on the surface of cancer cells at all disease stages of prostate cancer progression. In particular, radiolabeled PSMA-targeting small-molecule ligands such as the beta emitter ¹⁷⁷Lu-PSMA-617 have demonstrated promising early clinical response rates (2). Clinical data from individual patients indicate that alpha emitters may surpass beta radiation in efficacy and can overcome therapeutic resistance (3). Although beta emitters have currently not been clinically tested after alpha emitter therapy, the strong efficacy of alpha radiation might be explained by the high linear energy transfer of alpha emitters as well as their ability to induce clustered DNA DSBs. These are particularly difficult for cells to repair and the highly localized energy of alpha emitters make them particularly attractive for the treatment of small volume metastases (26). In contrast, beta emitters induce mainly single-strand DNA breaks and mechanisms of resistance applicable for these are not valid for alpha emitters as recently demonstrated in an in vitro cell line screen (27).

Actinium-225 is an alpha emitter that has been used widely in preclinical and clinical settings (1, 3, 4, 28). Despite the encouraging clinical response rates for the PSMA-targeting small-molecule ligand ²²⁵Ac-PSMA-617 in noncontrolled trials (4), its clinical use, however, is hindered by severe xerostomia, which becomes a dose-limiting toxicity due to strong uptake of the small molecules into the salivary glands (6). This xerostomia has been observed to be less pronounced when combining PSMA-617 with beta emitters (29) instead of alpha emitters (3, 6). Nevertheless, this accumulation is thought to be at least partially nonspecific, given the low-to-moderate PSMA expression in the salivary gland, and also due to the fact that PSMA-targeting antibody formats have shown only low salivary gland uptake (6–8). This observed difference in uptake suggests that antibody-based PSMA-targeted alpha therapy could be an attractive concept to complement therapy based on small-molecule ligands.

We herein present an antibody-based PSMA-TAT using thorium-227 (t_1/2 = 18.7 days), which, similarly to actinium-225 (t_1/2 = 10.7 days), has a half-life that is compatible with monoclonal antibodies. Indeed, two thorium-227 conjugates have entered early clinical trials for the treatment of hematologic malignancies and selected solid cancers [NCT02581878 for CD22-TTC (BAY1862864), NCT03507452 for MSLN-TTC (BAY2287411); refs. 12, 15].

Thorium-227 (t_1/2 = 18.7 days) decays first to radium-223 (t_1/2 = 10 days), and finally to stable lead-207 in seven decay steps with emission of five alpha and two beta particles, as well as gamma radiation, along the way. Thus, the first decay step generates radium-223, which in form of radium-223 dichloride has a well-documented safety profile and proven survival benefit in prostate cancer patients with bone metastases and no know visceral metastases (5). Radium-223 dichloride is the first approved alpha pharmaceutical for the treatment of cancer and thus supports the great promise for TAT in prostate cancer (5). The herein investigated PSMA-TTC (BAY 2315497) consists of a fully human IgG1 antibody (16, 17) covalently linked to a 3,2-HOPO chelator for complexation of the radionuclide. Therefore, it has the potential to be used also in patients with visceral metastases, due to the targeted delivery of the alpha-particle emitter to all PSMA-positive lesions.

We have shown here that PSMA-TTC can be produced with consistently high yields of the radiolabeled drug product. The product is stable over 48 hours, further facilitating centralized radiopharmacy production and distribution. In addition, the production of PSMA-TTC can be facilitated by the established manufacturing of the commercially available drug radium-223 chloride for clinical use as thorium-227 is the parent nuclide to radium-223.

In the studies presented herein, PSMA-TTC shows rapid and selective uptake as well as potent induction of DNA damage, cell-cycle arrest, and apoptosis in vitro. In vivo, PSMA-TTC demonstrates potent antitumor efficacy as measured by changes in tumor size and PSA levels in a number of prostate cancer xenograft models with clinically relevant PSMA expression levels, including the 22Rv1 model showing only moderate/heterogeneous expression of PSMA.

The prostate cancer xenograft models harbor various clinically observed resistance mechanisms such as mutation of the androgen receptor (KUCaP-1; refs. 19 and 30) or androgen-independent tumor growth through upregulation of different AR splice variants (22Rv1, ref. 31; LuCaP 86.2, ref. 20). Interestingly, the antitumor activity of PSMA-TTC is independent of these attributes. Marked in vivo efficacy was detected both in androgen-sensitive prostate cancer models (LNCaP and ST1273) and in models resistant to antiandrogens, including the second-generation AR antagonist enzalutamide (KUCaP-1). The efficacy of PSMA-TTC in the models mimicking CRPC is critical, as only few therapies currently exist for this advanced stage of the disease, despite the fact that patients inevitably develop resistance to androgen deprivation and AR antagonist therapy over time. Potent antitumor efficacy was also observed in a prostate cancer bone growth model mimicking the bone metastatic stage affecting up to 90% of late-stage mCRPC patients (22–24). In summary, these data exemplify the potential of PSMA-TTC as a candidate for TAT.

PSMA-TTC inhibits tumor growth at both single and multiple dosing, and neither its efficacy nor its biodistribution is affected by varying the total antibody dose from 0.14 up to 1.5 mg/kg. This would translate into a total antibody dose from approximately 10 to 100 mg for a 70-kg patient. However, the use of a total antibody dose of 5 mg/kg (corresponding approximately to 350 mg per patient) resulted in reduction in antitumor activity when compared with the lower total antibody doses in the 22Rv1 model that harbors moderate/heterogeneous PSMA expression. This indicates that potential receptor saturation may limit the efficacy of PSMA-TTC. Taken together, these findings support the clinical exploration of escalating both the total antibody dose and the radioactive dose at varying schedules to maximize the therapeutic window.

In the biodistribution studies presented, selective tumor uptake and tumor retention of radioactivity for >21 days were observed in mice after treatment with PSMA-TTC. This differs from radiotherapy based on small-molecule ligands where radioactivity in tumors strongly diminishes over 72 hours (32). Thus, antibody-based radionuclide therapy is compatible with the alpha emitter thorium-227 that has a long half-life of 18.7 days.

PSMA-TTC was found to be well tolerated in mice based on body weight monitoring, and the observed myelosuppression was dose dependent and showed signs of recovery. Clinically, myelosuppression is a common phenomenon when using IgG-based radiotherapeutics (8, 28). Antibody-based radioimmunotherapy (RIT) with beta emitters, namely, the CD20-targeting antibodies I-131-tositumomab and Y90-ibritumomab tiuxetan (33), are approved for the treatment of hematologic malignancies, as for these drugs the predicted myelosuppression has been shown to be manageable and due to the long-term response rates, a positive benefit–risk assessment has been demonstrated. However, in solid tumors, the observed myelosuppression (neutropenia and thrombocytopenia) has been shown to limit the therapeutic window as observed for a previously tested PSMA-targeted antibody-based radioimmunotherapy using the beta emitter lutetium-177 (8, 34, 35).

The myelosuppressive side effects of antibody-based RIT using beta emitters are partially attributed to “bystander” irradiation of normal tissues such as the bone marrow as a result of the long circulation time of the antibody and the long path length of the beta emitter. Accordingly, reports from early trials in hematologic malignancies show that antibody-based RIT using alpha emitters allows more efficient and selective cell killing compared with RIT using beta emitters delivered by monoclonal antibodies within the tolerated range. For example in acute myeloid leukemia, alpha emitter-based radiotherapy using the monoclonal CD33 antibody lintuzumab (with actinium-225 and bismuth-213) has been clinically tested and deemed favorable compared with beta emitters with greater efficacy within the tolerated range (26, 28, 33). However, also with ²²⁵Ac-lintuzumab, the main dose-limiting toxicity was prolonged myelosuppression and, thus, careful evaluation of dosing and scheduling is required.

The toxicological findings of myelosuppression by PSMA-TTC observed in mice have been confirmed in preclinical safety studies in monkeys (data not shown) that also enabled the first-in-man (FiM) studies. Careful dose escalation with relevant safety measures is, therefore, being carried out in humans to identify optimal dose of thorium-227 as well as total antibody. In addition, combination therapy with partners with nonoverlapping toxicity such as antihormonal agents or immune oncology drugs may provide an opportunity to further broaden the therapeutic window.

Whereas it can be assumed that the toxicity profiles of a PSMA ligand and a PSMA-targeting antibody differ due to the difference in the size of the molecule and hence, excretion route, the overall safety profiles of PSMA small ligands and the herein presented PSMA-TTC remain to be elucidated in controlled trials with long-term follow-up (36). Therefore, it can also be speculated that there is potential to combine antibody-based and small ligand-based PSMA-targeting radiotherapeutics due to their nonoverlapping binding sites at the PSMA target antigen and their nonoverlapping toxicities, and these combinations should be studied in the future.

PSMA-TTC demonstrates potent antitumor efficacy in a panel of cell and patient-derived models mimicking different stages of prostate cancer. The presented data support the clinical development of PSMA-TTC for the treatment of mCRPC patients and a clinical phase I trial has been initiated (NCT03724747).

S. Hammer is an employee/paid consultant for and holds ownership interest (including patents) in Bayer AG. U.B. Hagemann and S. Zitzmann-Kolbe are employees/paid consultants for Bayer AG. A. Larsen, C. Ellingsen, S. Geraudie, D. Grant, B. Indrevoll, and R. Smeets are employees/paid consultants for Bayer AS. O. von Ahsen is an employee/paid consultant for Bayer AG. A Kristian is an employee/paid consultant for Bayer AS. P. Lejeune is an employee/paid consultant for Bayer AG. H. Hennekes is an employee of and holds ownership interest in Bayer AG. J. Karlsson, R.M. Bjerke, and O.B. Ryan are employees/paid consultants for Bayer AS. A.S. Cuthbertson is an employee/paid consultant for and holds ownership interest (including patents) in Bayer AS. D. Mumberg is an employee/paid consultant for and holds ownership interest (including patents) in Bayer AG, and is an advisory board member/unpaid consultant for Keystone Symposia Scientific Advisory Board.

Conception and design: S. Hammer, U.B. Hagemann, S. Zitzmann-Kolbe, A. Larsen, A. Kristian, P. Lejeune, J. Karlsson, R.M. Bjerke, A.S. Cuthbertson

Development of methodology: S. Hammer, S. Zitzmann-Kolbe, A. Larsen, S. Geraudie, O. von Ahsen, A. Kristian, J. Karlsson

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Zitzmann-Kolbe, C. Ellingsen, S. Geraudie, B. Indrevoll, R. Smeets, O. von Ahsen, A. Kristian, J. Karlsson, R.M. Bjerke, O.B. Ryan

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Hammer, U.B. Hagemann, A. Larsen, C. Ellingsen, S. Geraudie, D. Grant, O. von Ahsen, A. Kristian, P. Lejeune, H. Hennekes, J. Karlsson, A.S. Cuthbertson

Writing, review, and/or revision of the manuscript: S. Hammer, U.B. Hagemann, S. Zitzmann-Kolbe, A. Larsen, C. Ellingsen, S. Geraudie, D. Grant, B. Indrevoll, R. Smeets, O. von Ahsen, A. Kristian, P. Lejeune, H. Hennekes, J. Karlsson, R.M. Bjerke, O.B. Ryan, A.S. Cuthbertson, D. Mumberg

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Hammer, U.B. Hagemann, A.S. Cuthbertson

Study supervision: S. Hammer, U.B. Hagemann, D. Grant, A.S. Cuthbertson, D. Mumberg

Other (planned and performed studies): S. Zitzmann-Kolbe

Aurexel Life Sciences Ltd. (www.aurexel.com) is acknowledged for editorial support funded by Bayer AG. We thank Pharmatest Services Ltd. (Finland), Biotest (Denmark), and Minerva Imaging (Denmark) for excellent technical and scientific support. We thank Manuela Steinbach, Stefan Stargard, Sandra Zickelbein, Elke Schmid, Volker Stickel, Martin Kohs, and Michael Reinhardt for excellent experimental assistance.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.