Development of 134Ce-PSMA-617 for Auger electron therapy and PET imaging of prostate cancer

Introduction: Despite the availability of optimal therapies, many prostate cancer patients progress to metastatic castrate-resistant prostate cancer (mCRPC), underscoring the pressing need for innovative treatment approaches [1]. Auger electron emitters, characterized by high linear energy transfer (4–26 keV/μm), are well-suited for addressing micrometastases when administered close to cancer cell DNA while minimizing damage to normal tissues [2]. We and others have recently proposed a ¹³⁴Ce/¹³⁴La (t_1/2=3.16d) pair reported as a potential surrogate for ²²⁵Ac [3-5], suitable for developing theranostic agents. ¹³⁴Ce decays to ¹³⁴La by emitting Auger electrons via electron capture. Herein, we evaluate the ¹³⁴Ce/La pair as an Auger electron therapy and PET imaging surrogate for prostate cancer using PSMA-617.

Methods: Radiolabeling was performed by incubating with ¹³⁴Ce in 0.1M NH₄OAc at 60 ^oC, 1 h, and ²²⁵Ac in 0.1M NH₄OAc at 75 ^oC, 1 h. Radiochemical yield (RCY) was determined by radio iTLC developed with 50mM EDTA, pH 5.5. In vitro, assays for ¹³⁴Ce/²²⁵Ac-PSMA-617 were performed in PC3PIP cells. The PET imaging and ex vivo biodistribution of ¹³⁴Ce-PSMA-617 were performed in PC3PIP and Flu tumor xenografts over 7 days.

Results: The radiolabeling was executed with optimized conditions, yielding radiochemical yields exceeding 95% with a ligand-to-metal ratio of 10:1 (Figure 1A, B). In vitro evaluations were conducted using both PSMA+ PC3-Pip and PSMA- PC3-Flu cell lines, with comparisons made to their respective ²²⁵Ac-PSMA-617 conjugates. Binding assays demonstrated nearly identical uptake for ¹³⁴Ce-PSMA-617 and ²²⁵Ac-PSMA-617 at the 1-hour time point, with a significant reduction when cells were pre-blocked with PSMA-2. The binding affinity for ¹³⁴Ce-PSMA-617 was determined to be 32.9± 3.1 nM, consistent with the binding affinities reported for ²²⁵Ac/¹⁷⁷Lu-PSMA conjugates [6]. Interestingly, membrane-bound internalization of ¹³⁴Ce-PSMA-617 revealed a higher percentage of activity associated with the membrane-bound fraction (69.1±2.7%) at 1 hour, compared to internalized activity (24.8±0.4%). However, after 24-hour incubation, internalized activity significantly increased to 54.5±1.0%, while membrane-bound activity decreased to 49.5±1.0%, similar trends observed for ²²⁵Ac-PSMA-617 (Figure 1C). In the a clonogenic cell survival assay, The PSMA positive PC3-Pip cells were sensitive to ¹³⁴Ce treatment, while PC3-flu cells were resistant (Figure 1D). MicroPET/CT imaging unveiled substantial PC3-Pip tumor accumulation at 4 hours post-injection, followed by a gradual decline in tumor uptake up to 72 hours post-injection. Conversely, PSMA-negative PC3-Flu tumors exhibited no detectable accumulation (Figure 1E). Ex-vivo biodistribution findings were consistent with the PET imaging results.

Conclusions: The in vitro assays for ¹³⁴Ce-PSMA-617 demonstrated excellent binding affinity and cytotoxicity towards PC3-Pip cells over negative control PC3-Flu cells. As expected given the chemical similarity of the probes, ¹³⁴Ce and ²²⁵Ac-PSMA-617 also demonstrated similar cell binding and internalization. The In vivo PET imaging and ex vivo biodistribution showed a high tumor uptake at 4 h post-injection. Overall, these results suggest that ¹³⁴Ce-PSMA-617 may serve as a potential Auger electron therapy agent for prostate cancer, and a theranostic pair for imaging alongside the therapeutic ²²⁵Ac-PSMA-617.

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