Treatment of Prostate Cancer with CD46-targeted 225Ac Alpha Particle Radioimmunotherapy

Abstract

Purpose: Radiopharmaceutical therapy is changing the standard of care in prostate cancer and other malignancies. We previously reported high CD46 expression in prostate cancer and developed an antibody-drug conjugate and immunoPET agent based on the YS5 antibody, which targets a tumor-selective CD46 epitope. Here, we present the preparation, preclinical efficacy, and toxicity evaluation of [225Ac]DOTA-YS5, a radioimmunotherapy agent based on the YS5 antibody.

Experimental design: [225Ac]DOTA-YS5 was developed, and its therapeutic efficiency was tested on cell-derived (22Rv1, DU145), and patient-derived (LTL-545, LTL484) prostate cancer xenograft models. Biodistribution studies were carried out on 22Rv1 tumor xenograft models to confirm the targeting efficacy. Toxicity analysis of the [225Ac]DOTA-YS5 was carried out on nu/nu mice to study short-term (acute) and long-term (chronic) toxicity.

Results: Biodistribution study shows that [225Ac]DOTA-YS5 agent delivers high levels of radiation to the tumor tissue (11.64% ± 1.37%ID/g, 28.58% ± 10.88%ID/g, 29.35% ± 7.76%ID/g, and 31.78% ± 5.89%ID/g at 24, 96, 168, and 408 hours, respectively), compared with the healthy organs. [225Ac]DOTA-YS5 suppressed tumor size and prolonged survival in cell line-derived and patient-derived xenograft models. Toxicity analysis revealed that the 0.5 μCi activity levels showed toxicity to the kidneys, likely due to redistribution of daughter isotope 213Bi.

Conclusions: [225Ac]DOTA-YS5 suppressed the growth of cell-derived and patient-derived xenografts, including prostate-specific membrane antigen-positive and prostate-specific membrane antigen-deficient models. Overall, this preclinical study confirms that [225Ac]DOTA-YS5 is a highly effective treatment and suggests feasibility for clinical translation of CD46-targeted radioligand therapy in prostate cancer.

Figure 1.

CD46 is a target for therapy in prostate cancer, including in PSMA-negative disease. A, Western blot analysis of the PDX tumors showing overexpression of CD46 in PSMA-positive as well as PSMA-negative tumors. B, MicroPET/CT image for detection of the LTL-484 model with the [⁸⁹Zr]DFO-YS5 probe at 4 days after injections (n = 4). C, Coronal microPET/CT images for LTL-545, LTL-484, LTL-331, and LTL-331R using ⁶⁸Ga-PSMA-11. Tumor regions are shown by yellow ellipses (n = 5). D, Comparison of the tumor uptake of ⁶⁸Ga-PSMA-11 in PDX model shows low to moderate uptake of ⁶⁸Ga-PSMA-11 in the indicated PDX models (data represented as mean ± SD, n = 5).

Figure 2.

Preparation and confirmation of retention of immunogenicity of the [²²⁵Ac]DOTA-YS5 by in vitro studies. A, Synthesis scheme of [²²⁵Ac]DOTA-YS5. B, iTLC plots of the crude reaction and the purified [²²⁵Ac]DOTA-YS5 showing 94.84% ± 8.1% (n = 3) purity. C, Magnetic beads assay showing 86.4% ± 1.5% binding of [²²⁵Ac]DOTA-YS5 denoting the preservation of immunogenicity of radiolabeled antibody. The addition of cold YS5 reduces binding to 17.4% ± 0.5%, demonstrating specificity (data represented as mean ± SD, n = 4). Cell-associated molecules of [²²⁵Ac]DOTA-YS5 in 22Rv1 (D) and DU145 (E) cells after the treatment for different timepoints. F, MTT assay showing dose-dependent reduction of cell viability from [²²⁵Ac]DOTA-YS5 treatment on 22Rv1 cells (IC₅₀: 80.0 ± 20.0 pCi/mL for [²²⁵Ac]DOTA-YS5 and 7.3 ± 0.9 nCi/mL for [²²⁵Ac]DOTA-IgG). G, Clonogenic survival of the 22Rv1 cells treated with [²²⁵Ac]DOTA-YS5 showed a dose-dependent decrease in the number of colonies after treatment (n = 3).

Figure 3.

Biodistribution of the [²²⁵Ac]DOTA-YS5 in 22Rv1 xenograft-bearing mice confirming high delivery of the [²²⁵Ac]DOTA-YS5 to tumor tissue. A, The distribution of [²²⁵Ac]DOTA-YS5 (represented as %ID/g, mean ± SD, n = 4) in organs collected from day 1 to day 17. B, Tumor to blood, tumor to muscle, and tumor to kidney ratios from the biodistribution studies indicate [²²⁵Ac]DOTA-YS5 clearance with simultaneous accumulation in tumor tissue. C, Gamma energy spectra showing accumulation of ²¹³Bi in kidneys. As compared with the equilibrium gamma energy spectra of [²²⁵Ac]DOTA-YS5, increased intensity of the ²¹³Bi was observed in kidney samples at 24 hours. D, iQID camera digital autoradiographic imaging for distribution of the [²²⁵Ac]DOTA-YS5 in 22Rv1 xenograft tumor and healthy tissues studied from day 1 to day 7 after injections. The distribution of the radioactivity in tumor tissue is heterogenous, whereas all the other organs show homogenous distribution.

Figure 4.

Histology and immunofluorescence imaging showing DNA damage after [²²⁵Ac]DOTA-YS5 treatment. H&E and phosphorylated γ-H2AX (p-γ.H2AX) staining confirms morphologic changes and p-γ.H2AX foci for [²²⁵Ac]DOTA-YS5 treatment for 7 days (A) and 14 days (B; n = 2).

Figure 5.

Chronic toxicity study of the [²²⁵Ac]DOTA-YS5 in nude mice (n = 4). A, The body weight measurements of the mice injected with the [²²⁵Ac]DOTA-YS5 show a gradual decrease with 0.5 μCi administration, whereas no significant body weight loss was seen in the 0.25 μCi dose or in the saline control group. B, Survival plot in the toxicity study. C, Liver and kidney function tests results showing increase in creatinine, blood urea nitrogen, and alkaline phosphatase. D, Blood cell counts confirming no significant difference in saline control versus [²²⁵Ac]DOTA-YS5–injected mice. E, Histologic H&E findings in the kidney of a mouse injected with 0.5 μCi activity level with diffuse parenchymal damage and occlusion of glomerular capillary loops by fibrin thrombi with glomerulosclerosis. F, Trichrome stain highlighting fibrin thrombi. G, Higher power images showing tubular injury in the mouse treated with [²²⁵Ac]DOTA-YS5. One-way ANOVA P values are indicated as *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6.

Antitumor activity of [²²⁵Ac]DOTA-YS5 in subcutaneous 22Rv1 and PDX tumor models. A, Effect of [²²⁵Ac]DOTA-YS5 in 22Rv1 tumor-bearing mice (n = 7). Tumor volume measurements demonstrated delayed 22Rv1 tumor growth in 0.25 and 0.5 μCi dose groups of [²²⁵Ac]DOTA-YS5. Average body weights of the mice indicated gradual body weight loss in the 0.5 μCi treatment group. Kaplan–Meier survival plot showing improved survival probability of the [²²⁵Ac]DOTA-YS5–treated 22Rv1 xenograft-bearing mice compared against control groups. B, Effect of [²²⁵Ac]DOTA-YS5 in LTL-545 PDX. Tumor volumes, average body weights, and overall survival in the animals administered with [²²⁵Ac]DOTA-YS5 at 0.03, 0.06, 0.125, and 0.25 μCi dose treatment (n = 5). C, Tumor size, overall survival, and body weight for the LTL-484 PDX mice treated with [²²⁵Ac]DOTA-YS5 (n = 4). Data represented as mean ± SD.