UC Berkeley

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.