Targeted nanotheranostic systems offer significant benefits due to the integration of diagnostic and therapeutic functionality, promoting personalized medicine. In recent years, prostate-specific membrane antigen (PSMA) has emerged as an ideal theranostic target, fueling multiple new drug approvals and changing the standard of care in prostate cancer (PCa). PSMA-targeted nanosystems such as self-assembled nanoparticles (NPs), liposomal structures, water-soluble polymers, dendrimers, and other macromolecules are under development for PCa theranostics due to their multifunctional sensing and therapeutic capabilities. Herein, we discuss the significance and up-to-date development of "PSMA-targeted nanocarrier systems for radioligand imaging and therapy of PCa". The review also highlights critical parameters for designing nanostructured radiopharmaceuticals for PCa, including radionuclides and their chelators, PSMA-targeting ligands, and the EPR effect. Finally, prospects and potential for clinical translation is discussed.

Central nervous system tumors have resisted effective chemotherapy because most therapeutics do not penetrate the blood-tumor-brain-barrier. Nanomedicines between  ~ 10 and 100 nm accumulate in many solid tumors by the enhanced permeability and retention effect, but it is controversial whether the effect can be exploited for treatment of brain tumors. PLX038A is a long-acting prodrug of the topoisomerase 1 inhibitor SN-38. It is composed of a 15 nm 4-arm 40 kDa PEG tethered to four SN-38 moieties by linkers that slowly cleave to release the SN-38. The prodrug was remarkably effective at suppressing growth of intracranial breast cancer and glioblastoma (GBM), significantly increasing the life span of mice harboring them. We addressed the important issue of whether the prodrug releases SN-38 systemically and then penetrates the brain to exert anti-tumor effects, or whether it directly penetrates the blood-tumor-brain-barrier and releases the SN-38 cargo within the tumor. We argue that the amount of SN-38 formed systemically is insufficient to inhibit the tumors, and show by PET imaging that a close surrogate of the 40 kDa PEG carrier in PLX038A accumulates and is retained in the GBM. We conclude that the prodrug penetrates the blood-tumor-brain-barrier, accumulates in the tumor microenvironment and releases its SN-38 cargo from within. Based on our results, we pose the provocative question as to whether the 40 kDa nanomolecule PEG carrier might serve as a Trojan horse to carry other drugs past the blood-tumor-brain-barrier and release them into brain tumors.

²²⁵Ac-targeted α-radiotherapy is a promising approach to treating malignancies, including prostate cancer. However, α-emitting isotopes are difficult to image because of low administered activities and a low fraction of suitable γ-emissions. The in vivo generator ¹³⁴Ce/¹³⁴La has been proposed as a potential PET imaging surrogate for the therapeutic nuclides ²²⁵Ac and ²²⁷Th. In this report, we detail efficient radiolabeling methods using the ²²⁵Ac-chelators DOTA and MACROPA. These methods were applied to radiolabeling of prostate cancer imaging agents, including PSMA-617 and MACROPA-PEG₄-YS5, for evaluation of their in vivo pharmacokinetic characteristics and comparison to the corresponding ²²⁵Ac analogs. Methods: Radiolabeling was performed by mixing DOTA/MACROPA chelates with ¹³⁴Ce/¹³⁴La in NH₄OAc, pH 8.0, at room temperature, and radiochemical yields were monitored by radio-thin-layer chromatography. In vivo biodistributions of ¹³⁴Ce-DOTA/MACROPA.NH₂ complexes were assayed through dynamic small-animal PET/CT imaging and ex vivo biodistribution studies over 1 h in healthy C57BL/6 mice, compared with free ¹³⁴CeCl₃ In vivo, preclinical imaging of ¹³⁴Ce-PSMA-617 and ¹³⁴Ce-MACROPA-PEG₄-YS5 was performed on 22Rv1 tumor-bearing male nu/nu-mice. Ex vivo biodistribution was performed for ¹³⁴Ce/²²⁵Ac-MACROPA-PEG₄-YS5 conjugates. Results: ¹³⁴Ce-MACROPA.NH₂ demonstrated near-quantitative labeling with 1:1 ligand-to-metal ratios at room temperature, whereas a 10:1 ligand-to-metal ratio and elevated temperatures were required for DOTA. Rapid urinary excretion and low liver and bone uptake were seen for ¹³⁴Ce/²²⁵Ac-DOTA/MACROPA. NH₂ conjugates in comparison to free ¹³⁴CeCl₃ confirmed high in vivo stability. An interesting observation during the radiolabeling of tumor-targeting vectors PSMA-617 and MACROPA-PEG₄-YS5-that the daughter ¹³⁴La was expelled from the chelate after the decay of parent ¹³⁴Ce-was confirmed through radio-thin-layer chromatography and reverse-phase high-performance liquid chromatography. Both conjugates, ¹³⁴Ce-PSMA-617 and ¹³⁴Ce-MACROPA-PEG₄-YS5, displayed tumor uptake in 22Rv1 tumor-bearing mice. The ex vivo biodistribution of ¹³⁴Ce-MACROPA.NH₂, ¹³⁴Ce-DOTA and ¹³⁴Ce-MACROPA-PEG₄-YS5 corroborated well with the respective ²²⁵Ac-conjugates. Conclusion: These results demonstrate the PET imaging potential for ¹³⁴Ce/¹³⁴La-labeled small-molecule and antibody agents. The similar ²²⁵Ac and ¹³⁴Ce/¹³⁴La-chemical and pharmacokinetic characteristics suggest that the ¹³⁴Ce/¹³⁴La pair may act as a PET imaging surrogate for ²²⁵Ac-based radioligand therapies.

Tumoral uptake of large-size nanoparticles is mediated by the enhanced permeability and retention (EPR) effect, with variable accumulation and heterogenous tumor tissue penetration depending on the tumor phenotype. The performance of nanocarriers via specific targeting has the potential to improve imaging contrast and therapeutic efficacy in vivo with increased deep tissue penetration. To address this hypothesis, we designed and synthesized prostate cancer-targeting starPEG nanocarriers (40 kDa, 15 nm), [⁸⁹Zr]PEG-(DFB)₃(ACUPA)₁ and [⁸⁹Zr]PEG-(DFB)₁(ACUPA)₃, with one or three prostate-specific membrane antigen (PSMA)-targeting ACUPA ligands. The in vitro PSMA binding affinity and in vivo pharmacokinetics of the targeted nanocarriers were compared with a nontargeted starPEG, [⁸⁹Zr]PEG-(DFB)₄, in PSMA+ PC3-Pip and PSMA- PC3-Flu cells, and xenografts. Increasing the number of ACUPA ligands improved the in vitro binding affinity of PEG-derived polymers to PC3-Pip cells. While both PSMA-targeted nanocarriers significantly improved tissue penetration in PC3-Pip tumors, the multivalent [⁸⁹Zr]PEG-(DFB)₁(ACUPA)₃ showed a remarkably higher PC3-Pip/blood ratio and background clearance. In contrast, the nontargeted [⁸⁹Zr]PEG-(DFB)₄ showed low EPR-mediated accumulation with poor tumor tissue penetration. Overall, ACUPA conjugated targeted starPEGs significantly improve tumor retention with deep tumor tissue penetration in low EPR PC3-Pip xenografts. These data suggest that PSMA targeting with multivalent ACUPA ligands may be a generally applicable strategy to increase nanocarrier delivery to prostate cancer. These targeted multivalent nanocarriers with high tumor binding and low healthy tissue retention could be employed in imaging and therapeutic applications.

Radiopharmaceutical therapy using α -emitting 225 Ac is an emerging treatment for patients with advanced metastatic cancers. Measurement of the spatial dose distribution in organs and tumors is needed to inform treatment dose prescription and reduce off-target toxicity, at not only organ but also sub-organ scales. Digital autoradiography with α -sensitive detection devices can measure radioactivity distributions at 20-40 μm resolution, but anatomical characterization is typically limited to 2D. We collected digital autoradiographs across whole tissues to generate 3D dose volumes and used them to evaluate the simultaneous tumor control and regional kidney dosimetry of a novel therapeutic radiopharmaceutical for prostate cancer, [²²⁵Ac]Ac-Macropa-PEG₄-YS5, in mice. 22Rv1 xenograft-bearing mice treated with 18.5 kBq of [²²⁵Ac]Ac-Macropa-PEG₄-YS5 were sacrificed at 24 h and 168 h post-injection for quantitative α -particle digital autoradiography and hematoxylin and eosin staining. Gamma-ray spectroscopy of biodistribution data was used to determine temporal dynamics and 213 Bi redistribution. Tumor control probability and sub-kidney dosimetry were assessed. Heterogeneous 225 Ac spatial distribution was observed in both tumors and kidneys. Tumor control was maintained despite heterogeneity if cold spots coincided with necrotic regions. 225 Ac dose-rate was highest in the cortex and renal vasculature. Extrapolation of tumor control suggested that kidney absorbed dose could be reduced by 41% while maintaining 90% TCP. The 3D dosimetry methods described allow for whole tumor and organ dose measurements following 225 Ac radiopharmaceutical therapy, which correlate to tumor control and toxicity outcomes.

Purpose

Radiopharmaceutical therapy is changing the standard of care in prostate cancer and other malignancies. We previously reported high CD46 expression in prostate cancer and developed an antibody-drug conjugate and immunoPET agent based on the YS5 antibody, which targets a tumor-selective CD46 epitope. Here, we present the preparation, preclinical efficacy, and toxicity evaluation of [225Ac]DOTA-YS5, a radioimmunotherapy agent based on the YS5 antibody.

Experimental design

[225Ac]DOTA-YS5 was developed, and its therapeutic efficiency was tested on cell-derived (22Rv1, DU145), and patient-derived (LTL-545, LTL484) prostate cancer xenograft models. Biodistribution studies were carried out on 22Rv1 tumor xenograft models to confirm the targeting efficacy. Toxicity analysis of the [225Ac]DOTA-YS5 was carried out on nu/nu mice to study short-term (acute) and long-term (chronic) toxicity.

Results

Biodistribution study shows that [225Ac]DOTA-YS5 agent delivers high levels of radiation to the tumor tissue (11.64% ± 1.37%ID/g, 28.58% ± 10.88%ID/g, 29.35% ± 7.76%ID/g, and 31.78% ± 5.89%ID/g at 24, 96, 168, and 408 hours, respectively), compared with the healthy organs. [225Ac]DOTA-YS5 suppressed tumor size and prolonged survival in cell line-derived and patient-derived xenograft models. Toxicity analysis revealed that the 0.5 μCi activity levels showed toxicity to the kidneys, likely due to redistribution of daughter isotope 213Bi.

Conclusions

[225Ac]DOTA-YS5 suppressed the growth of cell-derived and patient-derived xenografts, including prostate-specific membrane antigen-positive and prostate-specific membrane antigen-deficient models. Overall, this preclinical study confirms that [225Ac]DOTA-YS5 is a highly effective treatment and suggests feasibility for clinical translation of CD46-targeted radioligand therapy in prostate cancer.

Purpose

Multiple myeloma is a plasma cell malignancy with an unmet clinical need for improved imaging methods and therapeutics. Recently, we identified CD46 as an overexpressed therapeutic target in multiple myeloma and developed the antibody YS5, which targets a cancer-specific epitope on this protein. We further developed the CD46-targeting PET probe [89Zr]Zr-DFO-YS5 for imaging and [225Ac]Ac-DOTA-YS5 for radiopharmaceutical therapy of prostate cancer. These prior studies suggested the feasibility of the CD46 antigen as a theranostic target in multiple myeloma. Herein, we validate [89Zr]Zr-DFO-YS5 for immunoPET imaging and [225Ac]Ac-DOTA-YS5 for radiopharmaceutical therapy of multiple myeloma in murine models.

Experimental design

In vitro saturation binding was performed using the CD46 expressing MM.1S multiple myeloma cell line. ImmunoPET imaging using [89Zr]Zr-DFO-YS5 was performed in immunodeficient (NSG) mice bearing subcutaneous and systemic multiple myeloma xenografts. For radioligand therapy, [225Ac]Ac-DOTA-YS5 was prepared, and both dose escalation and fractionated dose treatment studies were performed in mice bearing MM1.S-Luc systemic xenografts. Tumor burden was analyzed using BLI, and body weight and overall survival were recorded to assess antitumor effect and toxicity.

Results

[89Zr]Zr-DFO-YS5 demonstrated high affinity for CD46 expressing MM.1S multiple myeloma cells (Kd = 16.3 nmol/L). In vitro assays in multiple myeloma cell lines demonstrated high binding, and bioinformatics analysis of human multiple myeloma samples revealed high CD46 expression. [89Zr]Zr-DFO-YS5 PET/CT specifically detected multiple myeloma lesions in a variety of models, with low uptake in controls, including CD46 knockout (KO) mice or multiple myeloma mice using a nontargeted antibody. In the MM.1S systemic model, localization of uptake on PET imaging correlated well with the luciferase expression from tumor cells. A treatment study using [225Ac]Ac-DOTA-YS5 in the MM.1S systemic model demonstrated a clear tumor volume and survival benefit in the treated groups.

Conclusions

Our study showed that the CD46-targeted probe [89Zr]Zr-DFO-YS5 can successfully image CD46-expressing multiple myeloma xenografts in murine models, and [225Ac]Ac-DOTA-YS5 can effectively inhibit the growth of multiple myeloma. These results demonstrate that CD46 is a promising theranostic target for multiple myeloma, with the potential for clinical translation.

Rationale: ²²⁵Ac, a long-lived α-emitter with a half-life of 9.92 days, has garnered significant attention as a therapeutic radionuclide when coupled with monoclonal antibodies and other targeting vectors. Nevertheless, its clinical utility has been hampered by potential off-target toxicity, a lack of optimized chelators for ²²⁵Ac, and limitations in radiolabeling methods. In a prior study evaluating the effectiveness of CD46-targeted radioimmunotherapy, we found great therapeutic efficacy but also significant toxicity at higher doses. To address these challenges, we have developed a radioimmunoconjugate called ²²⁵Ac-Macropa-PEG₄-YS5, incorporating a stable PEGylated linker to maximize tumoral uptake and increase tumor-to-background ratios. Our research demonstrates that this conjugate exhibits greater anti-tumor efficacy while minimizing toxicity in prostate cancer 22Rv1 tumors. Methods: We synthesized Macropa.NCS and Macropa-PEG_4/8-TFP esters and prepared Macropa-PEG_0/4/8-YS5 (with nearly ~1:1 ratio of macropa chelator to antibody YS5) as well as DOTA-YS5 conjugates. These conjugates were then radiolabeled with ²²⁵Ac in a 2 M NH₄OAc solution at 30 °C, followed by purification using YM30K centrifugal purification. Subsequently, we conducted biodistribution studies and evaluated antitumor activity in nude mice (nu/nu) bearing prostate 22Rv1 xenografts in both single-dose and fractionated dosing studies. Micro-PET imaging studies were performed with ¹³⁴Ce-Macropa-PEG_0/4/8-YS5 in 22Rv1 xenografts for 7 days. Toxicity studies were also performed in healthy athymic nude mice. Results: As expected, we achieved a >95% radiochemical yield when labeling Macropa-PEG_0/4/8-YS5 with ²²⁵Ac, regardless of the chelator ratios (ranging from 1 to 7.76 per YS5 antibody). The isolated yield exceeded 60% after purification. Such high conversions were not observed with the DOTA-YS5 conjugate, even at a higher ratio of 8.5 chelators per antibody (RCY of 83%, an isolated yield of 40%). Biodistribution analysis at 7 days post-injection revealed higher tumor uptake for the ²²⁵Ac-Macropa-PEG₄-YS5 (82.82 ± 38.27 %ID/g) compared to other conjugates, namely ²²⁵Ac-Macropa-PEG_0/8-YS5 (38.2 ± 14.4/36.39 ± 12.4 %ID/g) and ²²⁵Ac-DOTA-YS5 (29.35 ± 7.76 %ID/g). The PET Imaging of ¹³⁴Ce-Macropa-PEG_0/4/8-YS5 conjugates resulted in a high tumor uptake, and tumor to background ratios. In terms of antitumor activity, ²²⁵Ac-Macropa-PEG₄-YS5 exhibited a substantial response, leading to prolonged survival compared to ²²⁵Ac-DOTA-YS5, particularly when administered at 4.625 kBq doses, in single or fractionated dose regimens. Chronic toxicity studies observed mild to moderate renal toxicity at 4.625 and 9.25 kBq doses. Conclusions: Our study highlights the promise of ²²⁵Ac-Macropa-PEG₄-YS5 for targeted alpha particle therapy. The ²²⁵Ac-Macropa-PEG₄-YS5 conjugate demonstrates improved biodistribution, reduced off-target binding, and enhanced therapeutic efficacy, particularly at lower doses, compared to ²²⁵Ac-DOTA-YS5. Incorporating theranostic ¹³⁴Ce PET imaging further enhances the versatility of macropa-PEG conjugates, offering a more effective and safer approach to cancer treatment. Overall, this methodology has a high potential for broader clinical applications.