Evaluation of 225Ac/134Ce-Macropa-PEG4-YS5 as a novel theranostic pair for prostate cancer therapy

Introduction: ²²⁵Ac, a long-lived (t_1/2 = 9.92 d) α-emitter, has been widely investigated as a therapeutic radionuclide in combination with monoclonal antibodies in various cancer models including prostate cancer. However, potential off-target toxicity, lack of chelators optimized for ²²⁵Ac, and radiolabeling methods limit their clinical applicability. Recently in our lab, we have developed ²²⁵Ac-DOTA-YS5, employing a cancer selective, CD46 antibody, for treatment of prostate cancer. PEGylated antibodies have shown a higher blood clearance with improved tumor-to-background ratios compared to non-PEGylated antibody conjugates. Here we combine the use of novel linker technology with the high-affinity Macropa chelator to conjugate the therapeutic radioisotope ²²⁵Ac with the specific targeting antibody YS5 for the treatment of prostate cancer, to assess therapeutic efficacy and toxicity.

Methods: Macropa NCS and Macropa-PEG₄-TFP esters were synthesized by following prior literature. Macropa-PEG₈-TFP ester was synthesized in two steps. These intermediates were then conjugated with the YS5 antibody through incubation in a Na₂CO₃-NaHCO₃ buffer, pH=9.0, at 37°C for 2 h, followed by purification with a PD10 column. Radiolabeling of Macropa-PEG_0/4/8-YS5 conjugates was achieved by incubating with Ac-225 in 2M NH₄OAc, pH=5.8, at 30°C for 30 minutes, followed by YM30K centrifugal filtration. Ce-134 radiolabeling was performed in 1M NH₄OAc, pH=8.0, at 30°C for 30 min. Micro-PET imaging studies were conducted with ¹³⁴Ce-Macropa-PEG_0/4/8-YS5 in 22Rv1 xenografts over 7 days. Ex vivo biodistribution was analyzed at 1, 2, 4, and 7 days post-injection with a 0.5 µCi dose. Antitumor efficacy of ²²⁵Ac-Macropa-PEG₄-YS5 conjugate was evaluated in 22Rv1 xenografts with doses of 0.125 and 0.25 µCi, using fractionated treatment doses of 3x0.125 µCi (0, 10, 24 days).

Results: The study involved modifying the Macropa chelator by incorporating PEG linkers and coupling it to YS5 IgG. Non-specific bioconjugation resulted in low (1:1) and high (>2.5) chelator ratios per YS5 antibody. Regardless of the chelator ratio, >95% radiochemical yield was achieved when labeling Macropa-PEG_0/4/8-YS5 with 225Ac, with isolated yields >60% after purification. In contrast, DOTA-YS5 conjugate with 8.7 chelators per antibody showed lower conversion rates. MicroPET imaging of ¹³⁴Ce-Macropa-PEG_0/4/8-YS5 displayed high tumor uptake at 7 days post-injection in 22Rv1 tumor xenografts. Biodistribution at 7 days revealed higher tumor uptake for PEG₄ (82.82±38.27 %ID/g) compared to PEG₈ (38.15±14.41 %ID/g), PEG₀ (36.39±12.4 %ID/g), and DOTA (29.35±7.76 %ID/g) conjugates. Conjugates with higher chelator numbers exhibited lower tumor uptake (PEG₀: 18.5±7.2 %ID/g, PEG₄: 34.7±9.1 %ID/g, PEG₈: 18.51±5.55 %ID/g). These findings indicate that heavy modification of YS5 led to immunoreactivity loss, with PEG4 showing the highest tumor-to-background ratios. ²²⁵Ac-Macropa-PEG₄-YS5 exhibited high binding affinity, saturation binding constant (2.5±1.2 nM), and half-minimal inhibitory concentration (IC50) of 0.41±0.21 nCi/mL in 22Rv1 cells. Antitumor activity and overall survival for ²²⁵Ac-Macropa-PEG₄-YS5 (54 days, 0.125 µCi) significantly improved over ²²⁵Ac-DOTA-YS5 (38 days, P=0.0005), saline control (27 days, p<0.0001), with no difference noted for higher dose 0.25 µCi. A fractionated treatment study demonstrated improved anti-tumor effect and overall survival for ²²⁵Ac-Macropa-PEG₄-YS5 (75 days) versus ²²⁵Ac-DOTA-YS5 (50 days) and saline (17.5 days). Toxicology studies in mice showed mild to moderate kidney toxicity, with no damage to other organs. Comprehensive blood, liver, and kidney function tests revealed no signs of toxicity.

Conclusions: PEGylated ²²⁵Ac-Macropa-PEG₄-YS5 improves biodistribution, reduces off-target binding, and enhances efficacy compared to ²²⁵Ac-DOTA-YS5 in 22Rv1 prostate cancer, promising a generally applicable strategy for alpha radioimmunotherapy.

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