Targeted Radionuclide Therapy Using a Lutetium-177 Labeled Human Anti-CD133 Antibody

Targeted radionuclide therapy against cancer stem cell-specific markers, such as CD133, constitutes a promising strategy to eliminate resilient cancer stem cells for improved outcomes in refractory tumors. Here, we report the synthesis and evaluation of [¹⁷⁷Lu]Lu-DOTA-RW03, a CD133-targeted radioimmunotherapy.

A fully human, anti-CD133 antibody (RW03) was conjugated with DOTA-NHS and radiolabeled with lutetium-177 to yield [¹⁷⁷Lu]Lu-DOTA-RW03. Radioligand binding assays on [¹⁷⁷Lu]Lu-DOTA-RW03 were performed using CD133 expressing HT-29 cells to determine binding affinity and immunoreactive fraction. Immunodeficient mice (n = 15) bearing HT-29 tumors were divided into 4 cohorts to establish the biodistribution of [¹⁷⁷Lu]Lu-DOTA-RW03 at 24, 48, and 96 h post-injection (n = 5 per cohort). Additional biodistribution and SPECT imaging studies were performed to establish tumor specificity and dose-dependent tumor uptake. In a dose-escalation therapy study, HT-29 tumor bearing mice (n = 20) were treated with either 4.0 ± 0.1, 9.6 ± 0.1, or 14.1 ± 0.2 MBq of [¹⁷⁷Lu]Lu-DOTA-RW03 or a vehicle control (n = 5 mice per cohort). Tumors from the therapy study were processed ex vivo for immunohistochemical and histopathological analysis.

Radioimmunoconjugate [¹⁷⁷Lu]Lu-DOTA-RW03 (4.4 ± 0.1 DOTA per antibody) was isolated in 50 ± 10% radiochemical yield, 17-28 GBq/µmol molar activity, and in > 98% radiochemical purity. In vitro, the radiolabeled antibody exhibited excellent binding affinity (0.30 ± 0.03 nM) and > 75% immunoreactivity. The biodistribution of [¹⁷⁷Lu]Lu-DOTA-RW03 revealed notable tumor uptake (65 ± 5%ID/g, 96 h post-injection) and a favorable tumor-to-blood ratio (5:1, 96 h post-injection). In vivo antigen specificity was confirmed by a significant reduction (75%) in tumor uptake when [¹⁷⁷Lu]Lu-DOTA-RW03 was co-administered with a 200-fold molar excess of unlabeled RW03. The radioimmunoconjugate exhibited promising therapeutic efficacy in the treatment of CD133 expressing colorectal xenograft mouse model, with dose-dependent reductions in tumor growth rate and increased survival time. Histopathological and immunohistochemical analyses revealed elevated cell proliferation and extensive liquefactive necrosis at late stages into treatment, which provides an opportunity for multidosing and combination treatment strategies.

These findings underscore the potential of [¹⁷⁷Lu]Lu-DOTA-RW03 as an effective therapy through targeting CD133 expressing cancer cells.