New Frontiers in Cancer Treatment: Unveiling the Potential of 3p-C-DEPA Chelators for Actinium-225 Targeted Alpha Therapy

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Introduction

Targeted Alpha Therapy (TAT) has emerged as a groundbreaking strategy in the fight against various cancers. This innovative approach utilizes alpha-emitting radioisotopes, such as Actinium-225 ( $^{225} A c$ ), to precisely deliver potent radiation to tumor cells. Unlike conventional beta-minus particles, alpha particles boast high linear energy transfer (LET) and a short path length, allowing for highly localized and destructive effects on cancer cells while minimizing collateral damage to healthy surrounding tissue. The unique decay chain of $^{225} A c$ results in the emission of four alpha particles, contributing to its remarkable cytotoxic potential, and its relatively long half-life makes it suitable for combination with long-circulating biologics like monoclonal antibodies.

However, a key challenge in developing effective $^{225} A c$ -based radiopharmaceuticals is identifying suitable bifunctional chelators (BFCs)—molecules that can securely bind the radioactive isotope and simultaneously attach to targeting vectors, such as antibodies. Traditional chelators often fall short in terms of stability or radiolabeling efficiency, particularly under mild conditions suitable for sensitive biomolecules. This study investigates the potential of novel DEPA-based chelators to overcome these limitations, benchmarking them against established alternatives like DOTA and macropa.

Key Findings from the Paper

This research extensively characterized novel DEPA-based chelators for their potential in targeted alpha therapy with Actinium-225 ( $^{225} A c$ ). The study focused on three specific bifunctional chelators (BFCs): 3p-C-DEPA-NO2, 3p-C-DEPA-NCS, and 3p-C-DEPA-TFP-PEG4, synthesizing them with high yield and purity.

A significant finding was the excellent radiochemical conversion (RCC) achieved with $[^{225} A c] A c$ -3p-C-DEPA-NO2, showing high labeling efficiency (93.7% to 96.8%) even at room temperature within one hour. This is a considerable advantage, as many existing chelators like DOTA require elevated temperatures for efficient labeling, which can be detrimental to heat-sensitive biomolecules like antibodies. The stability of this $^{225} A c$ -labeled complex was also remarkable, with over 95% remaining intact in human serum after six days.

The researchers then moved to evaluate these chelators when conjugated to trastuzumab, a monoclonal antibody, comparing them to DOTA-trastuzumab and macropa-trastuzumab. All immunoconjugates demonstrated high purity (98%). For the radiolabeled immunoconjugates, $[^{225} A c] A c$ -3p-C-DEPA-trastuzumab and $[^{225} A c] A c$ -3p-C-DEPA-TFP-PEG4-trastuzumab showed high RCCs (94.6% and 93.5% respectively), comparable to the established macropa-trastuzumab (96.5%). In contrast, $[^{225} A c] A c$ -DOTA-trastuzumab had a lower RCC of 80.9%, necessitating purification.

The in vitro stability of the radioimmunoconjugates in PBS revealed crucial differences. While $[^{225} A c] A c$ -3p-C-DEPA-trastuzumab showed limited stability (only 48.3% intact after 10 days, likely due to a specific linker instability), $[^{225} A c] A c$ -3p-C-DEPA-TFP-PEG4-trastuzumab exhibited superior stability, maintaining over 90% integrity after 10 days. This performance was comparable to $[^{225} A c] A c$ -macropa-trastuzumab (81.9% intact) and significantly better than $[^{225} A c] A c$ -DOTA-trastuzumab (60.1% intact).

In vivo biodistribution studies in healthy mice demonstrated similar organ distribution profiles for $[^{225} A c] A c$ -3p-C-DEPA-trastuzumab, $[^{225} A c] A c$ -DOTA-trastuzumab, and $[^{225} A c] A c$ -macropa-trastuzumab across most organs. Notably, $[^{225} A c] A c$ -3p-C-DEPA-trastuzumab and $[^{225} A c] A c$ -macropa-trastuzumab showed significantly lower liver uptake compared to $[^{225} A c] A c$ -DOTA-trastuzumab. High blood retention was observed for all constructs, indicating long circulation times for the intact antibody conjugates. This lower liver uptake is a key advantage, as it suggests reduced off-target radiotoxicity, a known concern with free $^{225} A c$ .

Role of ichorbio's antibodies

ichorbio's antibodies played a vital role in this study, serving as the targeting vector for the radiopharmaceutical conjugates. Specifically, the researchers utilized:

Trastuzumab (catalogue number: ICH4013)

Trastuzumab, a research-grade monoclonal antibody, was conjugated with the bifunctional chelators (3p-C-DEPA-NCS, 3p-C-DEPA-TFP-PEG4, DOTA-NCS, and macropa-NCS) to form immunoconjugates. These conjugates were then radiolabeled with Actinium-225 and evaluated for their radiochemical properties, in vitro stability, and in vivo biodistribution. Trastuzumab’s well-established use in targeted therapies made it an ideal candidate to assess the performance of the novel chelators in a clinically relevant context.

Implications and Future Directions

The findings of this study carry significant implications for the development of next-generation $^{225} A c$ -based radiopharmaceuticals. The 3p-C-DEPA-TFP-PEG4 chelator stands out as a highly promising candidate due to its exceptional radiolabeling efficiency under mild conditions and superior in vitro stability, particularly its resilience to degradation. The low liver uptake observed with the 3p-C-DEPA-based conjugates in vivo is particularly important, as it suggests a reduced risk of hepatotoxicity, a significant concern with other $^{225} A c$ chelators.

This research indicates that 3p-C-DEPA-TFP-PEG4 could be a strong alternative to current $^{225} A c$ bifunctional chelators, potentially accelerating the clinical translation of novel targeted alpha therapies. Further validation of its therapeutic potential through comprehensive preclinical studies, especially in tumor-bearing mouse models, is the logical next step.

Suggested Future Experiments

Based on these promising results, several key future experiments are warranted:

Pharmacokinetic Studies in Tumor-Bearing Models: While biodistribution in healthy mice was evaluated, it is crucial to assess the pharmacokinetics and tumor-targeting efficiency of $[^{225} A c] A c$ -3p-C-DEPA-TFP-PEG4-trastuzumab in tumor-bearing mouse models. This will provide direct evidence of its ability to selectively accumulate in tumors.
Therapeutic Efficacy Studies: Conduct preclinical studies to evaluate the therapeutic efficacy of the new conjugates in relevant cancer models. This would involve assessing tumor growth inhibition, survival rates, and potential for complete remission.
Long-Term In Vivo Stability: While 48-hour biodistribution showed good in vivo performance, longer-term in vivo stability studies are needed to confirm the kinetic inertness of the $^{225} A c$ -3p-C-DEPA-TFP-PEG4 complex over extended periods, especially given the decay chain of $^{225} A c$ and potential for daughter radionuclide redistribution.
Comparison of Therapeutic Indices: A head-to-head comparison of the therapeutic window (efficacy vs. toxicity) of $[^{225} A c] A c$ -3p-C-DEPA-TFP-PEG4-trastuzumab with established chelators like macropa and DOTA in tumor models would be highly valuable.
Exploration of Other Targeting Vectors: Investigate the conjugation of 3p-C-DEPA-TFP-PEG4 to other types of targeting vectors (e.g., peptides, small molecules) to expand its applicability beyond monoclonal antibodies.
Detailed Mechanism of Stability for 3p-C-DEPA-TFP-PEG4: Further investigate the specific chemical reasons behind the superior stability of the PEGylated TFP derivative, especially its resistance to radiolysis in the presence of chloride ions, to inform the rational design of even more robust chelators.

Conclusion

This study successfully characterizes 3p-C-DEPA-based chelators, particularly 3p-C-DEPA-TFP-PEG4, as highly promising candidates for Actinium-225 targeted alpha therapy. Its remarkable radiolabeling efficiency, exceptional in vitro stability, and favorable biodistribution profile with low liver uptake position it as a strong contender to advance the field of nuclear medicine. These findings pave the way for future preclinical and clinical investigations, ultimately aiming to bring more effective and safer targeted alpha therapies to cancer patients.

Reference

Ketchemen, et al. Preclinical characterization of 3p-C-DEPA-NCS and 3p-C-DEPA-TFP-PEG4 as potential Actinium-225 bifunctional chelators using DOTA-NCS and macropa-NCS as benchmarks., 17 June 2025, PREPRINT (Version 1) available at Research Square [https://doi.org/10.21203/rs.3.rs-6813431/v1]