Development of CD46 targeted alpha theranostics in prostate cancer using 134Ce/225Ac-Macropa-PEG4-YS5

Abstract

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.

Keywords: PEG linkers; YS5 antibody; actinium-225; cerium-134; macropa; targeted alpha therapy; theranostics.

Figure 1

Synthesis and radiolabeling of Macropa-PEG_0/4/8-YS5 conjugates A) Bioconjugation of YS5 with Macropa-NCS and Macropa-PEG_4/8-TFP ester followed by radiolabeling with ²²⁵Ac(NO₃)₃ i) 0.1 M Na₂CO₃-Na₂HCO₃ buffer, pH = 9.0, 37 ^oC, 2 h followed by PD10 column purification and ii) ²²⁵Ac(NO₃)₃ in 0.2 M HCl, 150 mg/mL L-Ascorbic acid, 2 M NH₄OAc, pH = 5.8 followed by centrifugal filtration. B) Structures of Macropa-PEG_0/4/8-YS5 C) Radiochemical yields for ²²⁵Ac-Macropa-PEG₀-YS5, ²²⁵Ac-Macropa-PEG₄-YS5, ²²⁵Ac-Macropa-PEG₈-YS5 and ²²⁵Ac-DOTA-YS5 (In the figure legend denoted as a PEG₀, PEG₄, PEG₈, and DOTA). n = 2 reactions for PEG₀, PEG₄ and n = 1 reactions for PEG₈, DOTA.

Figure 2

²²⁵Ac-Macropa-PEG₄-YS5 demonstrates increased tumor uptake with improved tumor-to-background ratios and clearance profiles compared to other conjugates. A) Tumor uptakes from the ex-vivo biodistribution analysis of ²²⁵Ac-Macropa-PEG_0/4/8-YS5 and ²²⁵Ac-DOTA-YS5 in 22Rv1 xenografts after tail vein injection at day 1, 2, 4, and 7. B) Tumor to blood C) Tumor to muscle D) Tumor to kidney E) Tumor to liver ratios derived from ex vivo biodistribution analysis. F) Metabolic analysis of ²²⁵Ac-Macropa-PEG_0/4/8-YS5 and ²²⁵Ac-DOTA-YS5 in healthy nude mice over 7 days, %ID in urine. ²²⁵Ac-DOTA-YS5 biodistribution data was reproduced. Adapted with permission from , copyright 2023 American Association for Cancer Research.

Figure 3

µPET/CT imaging of ¹³⁴Ce-Macropa-YS5 agents as a theranostic pair for ²²⁵Ac therapy. Coronal µPET/CT images of mouse-bearing subcutaneous 22Rv1 xenografts at days 1, 2, 4, and 7 after administering the respective radio conjugates by tail vein demonstrate increased tumoral uptake over time. A) ¹³⁴Ce-Macropa-PEG₀-YS5. B) ¹³⁴Ce-Macropa-PEG₄-YS5. C) ¹³⁴Ce-Macropa-PEG₈-YS5. D) Ex vivo biodistribution after 7 days of PET Imaging (n = 4 mice for each group). The respective maximum intensity projection µPET/CT images are shown in Figure S25 in the supplementary appendix.

Figure 4

²²⁵Ac-Macropa-PEG₄-YS5 shows a high binding affinity for 22Rv1 cells, efficient cell killing, and elicits a robust DNA damage response which correlates with radiopharmaceutical uptake in vivo. A) Saturation binding assay curve of ²²⁵Ac-Macropa-PEG₄-YS5 in 22Rv1 cells. B) Activity-dependent cell death with ²²⁵Ac-Macropa-PEG₄-YS5; improved cell killing seen in ²²⁵Ac-Macropa-PEG₄-YS5 vs ²²⁵Ac-DOTA-YS5. C) Correlation of histology (H&E), immunofluorescence (phospho-γH2AX), and digital autoradiographic images after treatment with ²²⁵Ac-Macropa-PEG₄-YS5 over 7 days (n = 3). Data represented as Mean ± SD (n = 3).

Figure 5

A) Schematic illustration of single-dose treatment for ²²⁵Ac-Macropa-PEG₄-YS5 and ²²⁵Ac-DOTA-YS5 in subcutaneous 22Rv1 xenografts with 4.625 and 9.25 kBq doses. B) Average tumor volumes after the treatment. C) Percent survival as a function of time for the treatment and vehicle cohorts d) Normalized body weights. E) The median survival days and statistical analysis for the single treatment group (n = 8 per cohort), **p = 0.0052, ***p = 0.0005, ****p < 0.0001.

Figure 6

²²⁵Ac-Macropa-PEG₄-YS5 causes improved therapeutic outcomes compared against ²²⁵Ac-DOTA-YS5 in a fractionated dosing study. A) Schematic illustration for fractionated treatment study of ²²⁵Ac-Macropa-PEG₄-YS5 and ²²⁵Ac-DOTA-YS5 with a 3 x 4.625 kBq dose in 22Rv1 xenografts. B) Average tumor volumes, C) body weight, and D) Percent survival as a function of time for the treatment and vehicle cohorts. E) The median survival days and statistical analysis for the fractionation treatment group (n = 6 for saline group, n = 7 for ²²⁵Ac-Macropa-PEG₄-YS5 and ²²⁵Ac-DOTA-YS5 group. F) Micro PET/CT imaging using ⁸⁹Zr-DFO-YS5 in the group previously treated with ²²⁵Ac-Macropa-PEG₄-YS5 (3 x 4.625 kBq) in 22Rv1 xenografts. Coronal micro-PET/CT images were obtained on day 4 post-injection (n = 2, group 1) after the end of the study. G) Histology and immunofluorescence imaging of group 2 mice showing persistent CD46 expression in tumors following 3 x 4.625 kBq administrations of ²²⁵Ac-Macropa-PEG₄-YS5 (n = 3, group 2). ****p < 0.0001, undefined: not reached endpoint.