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[225Ac]Ac- and [111In]In-DOTA-trastuzumab theranostic pair: cellular dosimetry and cytotoxicity in vitro and tumour and normal tissue uptake in vivo in NRG mice with HER2-positive human breast cancer xenografts


Misaki Kondo, Zhongli Cai, Conrad Chan, Nubaira Forkan, and Raymond M. Reilly

EJNMMI Radiopharm Chem, 2023


Trastuzumab (Herceptin) has improved the outcome for patients with HER2-positive breast cancer (BC) but brain metastases (BM) remain a challenge due to poor uptake of trastuzumab into the brain. Radioimmunotherapy (RIT) with trastuzumab labeled with α-particle emitting, 225Ac may overcome this challenge by increasing the cytotoxic potency on HER2-positive BC cells. Our first aim was to synthesize and characterize [111In]In-DOTA-trastuzumab and [225Ac]Ac-DOTA-trastuzumab as a theranostic pair for imaging and RIT of HER2-positive BC, respectively. A second aim was to estimate the cellular dosimetry of [225Ac]Ac-DOTA-trastuzumab and determine its cytotoxicity in vitro on HER2-positive BC cells. A third aim was to study the tumour and normal tissue uptake of [225Ac]Ac-DOTA-trastuzumab using [111In]In-DOTA-trastuzumab as a radiotracer in vivo in NRG mice with s.c. 164/8-1B/H2N.luc+ human BC tumours that metastasize to the brain.


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 (KD = 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.


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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