Preclinical Evaluation of 225Ac-Labeled Single-Domain Antibody for the Treatment of HER2pos Cancer

Abstract

Human epidermal growth factor receptor type 2 (HER2) is overexpressed in various cancers; thus, HER2-targeting single-domain antibodies (sdAb) could offer a useful platform for radioimmunotherapy. In this study, we optimized the labeling of an anti-HER2-sdAb with the α-particle-emitter 225Ac through a DOTA-derivative. The formed radioconjugate was tested for binding affinity, specificity and internalization properties, whereas cytotoxicity was evaluated by clonogenic and DNA double-strand-breaks assays. Biodistribution studies were performed in mice bearing subcutaneous HER2pos tumors to estimate absorbed doses delivered to organs and tissues. Therapeutic efficacy and potential toxicity were assessed in HER2pos intraperitoneal ovarian cancer model and in healthy C57Bl/6 mice. [225Ac]Ac-DOTA-2Rs15d exhibited specific cell uptake and cell-killing capacity in HER2pos cells (EC50 = 3.9 ± 1.1 kBq/mL). Uptake in HER2pos lesions peaked at 3 hours (9.64 ± 1.69% IA/g), with very low accumulation in other organs (<1% IA/g) except for kidneys (11.69 ± 1.10% IA/g). α-camera imaging presented homogeneous uptake of radioactivity in tumors, although heterogeneous in kidneys, with a higher signal density in cortex versus medulla. In mice with HER2pos disseminated tumors, repeated administration of [225Ac]Ac-DOTA-2Rs15d significantly prolonged survival (143 days) compared to control groups (56 and 61 days) and to the group treated with HER2-targeting mAb trastuzumab (100 days). Histopathologic evaluation revealed signs of kidney toxicity after repeated administration of [225Ac]Ac-DOTA-2Rs15d. [225Ac]Ac-DOTA-2Rs15d efficiently targeted HER2pos cells and was effective in treatment of intraperitoneal disseminated tumors, both alone and as an add-on combination with trastuzumab, albeit with substantial signs of inflammation in kidneys. This study warrants further development of [225Ac]Ac-DOTA-2Rs15d.

Figure 1.

Cytotoxic activity of (i) [²²⁵Ac]Ac-DOTA-2Rs15d alone or (ii) blocked with cold 2Rs15d; and (iii) nontargeting [²²⁵Ac]Ac-DOTA-R3B23 control probe on SKOV-3 cells determined via clonogenic (A and B) and DNA double-strand break (C and D) assays. Representative images (A) and SFs (B) of SKOV-3 cells from clonogenic assay. C, The average number of γH2AX-foci quantified per cell; NT - control cells nontreated with radioactivity. D, Representative images of detected γH2AX-foci in treated SKOV-3 cells.

Figure 2.

In vivo specificity of [²²⁵Ac]Ac-DOTA-2Rs15d alone or blocked with excess of cold 2Rs15d, and nontargeting [²²⁵Ac]Ac-DOTA-R3B23 after 1 hour p.i. in subcutaneous SKOV-3 tumor-xenografted mice (A). *, P < 0.00015; **, P < 0.00004; ***, P < 0.000003. B, Time-dependent biodistribution of [²²⁵Ac]Ac-DOTA-2Rs15d in a subcutaneous SKOV-3 tumor-xenografted mouse model. C–F, Dose-escalation study of [²²⁵Ac]Ac-DOTA-2Rs15d in healthy C57Bl/6 mice with Kaplan–Meier survival plots (C) and relative body weight (D) in various groups of treated mice. Histopathologic analysis of kidney's sections from mice receiving only vehicle solution 0.9% NaCl (E) and 19 kBq of [²²⁵Ac]Ac-DOTA-2Rs15d (F).

Figure 3.

α-camera imaging of [²²⁵Ac]Ac-DOTA-2Rs15d activity distribution in cryo-sectioned HER2^pos tumors (A) and kidneys (B) of subcutaneous SKOV-3 xenografts. All images were taken at 1, 4, and 24 hours p.i. C, Histogram of the activity distribution of an α-camera image of kidneys 4 hours p.i. derived from ROIs encompassing the whole kidney area, including both cortex and medulla. D, Multi-term gaussian plots fitted to the histogram data of cortex ROI. Homogeneity of cortex (E) and tumor (F) was assessed by plots where the total signal fraction is displayed as a function of the total area fraction. A completely uniform ROI corresponds to a linear function (depicted as a dashed line) and an area under the curve (AUC) of 0.5.

Figure 4.

Therapy with [²²⁵Ac]Ac-DOTA-2Rs15d improves survival of mice bearing disseminated intraperitoneal SKOV3.IP1 tumors. Treatment schedules (A) and Kaplan–Meier survival plots (B) together with relative body weight (C) in various groups of treated mice. D, Calculated absorbed doses (Gy), without considering of RBE factor 5 for α-particles, in normal organs and tumor for one single therapeutic injection 85 kBq and total therapeutic activity 255 kBq of [²²⁵Ac]Ac-DOTA-2Rs15d. H&E sections of kidneys from mice receiving 0.9% NaCl (E), one single dose (F) and three injections (G) of [²²⁵Ac]Ac-DOTA-2Rs15d.