[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

Background: 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 alpha-particle emitting, Ac-225 may overcome this challenge by increasing the cytotoxic potency on HER2-positive BC cells. Our first aim was to synthesize and characterize [In-111]In-DOTA-trastuzumab and [Ac-225]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 [Ac-225]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 [Ac-225]Ac-DOTA-trastuzumab using [In-111]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.Results: Trastuzumab was conjugated to 12.7 +/- 1.2 DOTA chelators and labeled with In-111 or Ac-225. [In-111]In-DOTA-trastuzumab exhibited high affinity specific binding to HER2-positive SK-BR-3 human BC cells (K-D = 1.2 +/- 0.3 x 10(-8) mol/L). Treatment with [Ac-225]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/g. No surviving colonies were noted at SA = 1.10 kBq/mu g or 1.665 kBq/mu g. Multiple DNA double-strand breaks (DSBs) were detected in SK-BR-3 cells exposed to [Ac-225]Ac-DOTA-trastuzumab by gamma-H2AX immunofluorescence microscopy. The time-integrated activity of [In-111]In-DOTA-trastuzumab in SK-BR-3 cells was measured and used to estimate the absorbed doses from [Ac-225]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 (D-10) was 1.10 Gy. Based on the D-10 reported for gamma-irradiation of SK-BR-3 cells, we estimate that the relative biological effectiveness of the alpha-particles emitted by Ac-225 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 [In-111]In-DOTA-trastuzumab and [Ac-225]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 [In-111]In-DOTA-trastuzumab.Conclusion: We conclude that [Ac-225]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 [In-111]In-DOTA-trastuzumab. These results are promising for combining [In-111]In-DOTA-trastuzumab and [Ac-225]Ac-DOTA-trastuzumab as a theranostic pair for imaging and RIT of HER2-positive BC.