Development of CD46 targeted alpha theranostics in prostate cancer using Ce/Ac-Macropa-PEG-YS5.

Ac, 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 Ac, 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 Ac-Macropa-PEG-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. We synthesized Macropa.NCS and Macropa-PEG-TFP esters and prepared Macropa-PEG-YS5 (with nearly ~1:1 ratio of macropa chelator to antibody YS5) as well as DOTA-YS5 conjugates. These conjugates were then radiolabeled with Ac in a 2 M NHOAc 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 Ce-Macropa-PEG-YS5 in 22Rv1 xenografts for 7 days. Toxicity studies were also performed in healthy athymic nude mice. As expected, we achieved a >95% radiochemical yield when labeling Macropa-PEG-YS5 with Ac, 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 Ac-Macropa-PEG-YS5 (82.82 ± 38.27 %ID/g) compared to other conjugates, namely Ac-Macropa-PEG-YS5 (38.2 ± 14.4/36.39 ± 12.4 %ID/g) and Ac-DOTA-YS5 (29.35 ± 7.76 %ID/g). The PET Imaging of Ce-Macropa-PEG-YS5 conjugates resulted in a high tumor uptake, and tumor to background ratios. In terms of antitumor activity, Ac-Macropa-PEG-YS5 exhibited a substantial response, leading to prolonged survival compared to Ac-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. Our study highlights the promise of Ac-Macropa-PEG-YS5 for targeted alpha particle therapy. The Ac-Macropa-PEG-YS5 conjugate demonstrates improved biodistribution, reduced off-target binding, and enhanced therapeutic efficacy, particularly at lower doses, compared to Ac-DOTA-YS5. Incorporating theranostic Ce 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.