Results

Trastuzumab was conjugated to 12.7 ± 1.2 DOTA chelators and labeled with ¹¹¹In or ²²⁵Ac. [¹¹¹In]In-DOTA-trastuzumab exhibited high affinity specific binding to HER2-positive SK-BR-3 human BC cells (K_D = 1.2 ± 0.3 × 10^–8 mol/L). Treatment with [²²⁵Ac]Ac-DOTA-trastuzumab decreased the surviving fraction (SF) of SK-BR-3 cells dependent on the specific activity (SA) with SF < 0.001 at SA = 0.74 kBq/µg. No surviving colonies were noted at SA = 1.10 kBq/µg or 1.665 kBq/µg. Multiple DNA double-strand breaks (DSBs) were detected in SK-BR-3 cells exposed to [²²⁵Ac]Ac-DOTA-trastuzumab by γ-H2AX immunofluorescence microscopy. The time-integrated activity of [¹¹¹In]In-DOTA-trastuzumab in SK-BR-3 cells was measured and used to estimate the absorbed doses from [²²⁵Ac]Ac-DOTA-trastuzumab by Monte Carlo N-Particle simulation for correlation with the SF. The dose required to decrease the SF of SK-BR-3 cells to 0.10 (D₁₀) was 1.10 Gy. Based on the D₁₀ reported for γ-irradiation of SK-BR-3 cells, we estimate that the relative biological effectiveness of the α-particles emitted by ²²⁵Ac is 4.4. Biodistribution studies in NRG mice with s.c. 164/8-1B/H2N.luc⁺ human BC tumours at 48 h post-coinjection of [¹¹¹In]In-DOTA-trastuzumab and [²²⁵Ac]Ac-DOTA-trastuzumab revealed HER2-specific tumour uptake (10.6 ± 0.6% ID/g) but spleen uptake was high (28.9 ± 7.4% ID/g). Tumours were well-visualized by SPECT/CT imaging using [¹¹¹In]In-DOTA-trastuzumab.

Conclusion

We conclude that [²²⁵Ac]Ac-DOTA-trastuzumab exhibited potent and HER2-specific cytotoxicity on SK-BR-3 cells in vitro and HER2-specific uptake in s.c. 164/8-1B/H2N.luc⁺ human BC tumours in NRG mice, and these tumours were imaged by SPECT/CT with [¹¹¹In]In-DOTA-trastuzumab. These results are promising for combining [¹¹¹In]In-DOTA-trastuzumab and [²²⁵Ac]Ac-DOTA-trastuzumab as a theranostic pair for imaging and RIT of HER2-positive BC.

Results from nanoScan® SPECT/CT

• ¹¹¹In was used to measure the tumour and normal tissue uptake of the RICs due to the very low amount (4 kBq) of ²²⁵Ac that could be safely injected, assuming that coadministered ¹¹¹In and ²²⁵Ac-labeled RICs distribute equivalently
• The tumour uptake of [¹¹¹In]In-DOTA-trastuzumab was 2.5-fold significantly greater than irrelevant [¹¹¹In]In-DOTA-IgG₁ (10.6 ± 0.6% ID/g vs. 4.3 ± 0.7% ID/g respectively, P < 0.0001)
• Tumours were well-visualized by SPECT/CT in mice at 48 h post co-injection of [¹¹¹In]In-DOTA-trastuzumab and [²²⁵Ac]Ac-DOTA-trastuzumab, but not in mice co-injected with [¹¹¹In]In-DOTA-IgG₁ and [²²⁵Ac]Ac-DOTA-IgG₁

Fig. 5 a Tumour and normal tissue uptake at 48 h p.i. of [¹¹¹In]In-DOTA-trastuzumab (7–8 MBq) coinjected with [²²⁵Ac]Ac-DOTA-trastuzumab (4 kBq; total mass dose = 40 µg) in NRG mice with s.c. HER2-positive 164/8-1B/H2N.luc + human BC tumours. B: blood, Br: brain, H: heart, Lu: lungs, K: kidneys, Pn: pancreas, Sp: spleen, L: liver, S: stomach, I: intestine, Sk: skin, M: muscle, Bo: bone. Tissue activity was quantified by γ-counting of ¹¹¹In. Values shown are the mean ± SD (n = 4–5). Significant differences (*P < 0.05) are indicated by asterisks. b Representative SPECT/CT images of tumour-bearing NRG mice at 48 h p.i. of [¹¹¹In]In-DOTA-trastuzumab coinjected with [²²⁵Ac]Ac-DOTA-trastuzumab (left image) or [¹¹¹In]In-DOTA-IgG₁ coinjected with [²²⁵Ac]Ac-DOTA-IgG₁ (right image). T: tumour, Sp: spleen. Scale for SPECT/CT images shows the percent injected dose/g (%ID/g). Also shown in grayscale is the scale for the CT image

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