Preclinical Comparison of [<sup>111</sup>In]In- and\n[<sup>225</sup>Ac]Ac-DOTA-Trastuzumab IgG, F(ab′)<sub>2</sub> and Fab for Theranostic SPECT/CT Imaging and α‑Particle\nRadioimmunotherapy of HER2-Positive Human

Radioimmunotherapy\n(RIT) with α-particle-emitting, <sup>225</sup>Ac complexed to\ntrastuzumab may offer an alternative treatment for\npatients who progress on HER2-targeted therapies. Moreover, RIT with\n[<sup>225</sup>Ac]Ac-DOTA-trastuzumab could be combined with SPECT/CT\nimaging with [<sup>111</sup>In]In-DOTA-trastuzumab in a theranostic\napproach. In this study, we compared DOTA-conjugated trastuzumab IgG,\nF(ab')<sub>2</sub> or Fab complexed to <sup>111</sup>In or <sup>225</sup>Ac for SPECT/CT imaging and α-particle RIT of subcutaneous\n(s.c.) HER2-positive 164/8-1B/H2N.luc<sup>+</sup> human BC tumors\nin NRG mice. SPECT/CT imaging and tumor and normal tissue uptake were\ncompared in NRG or NOD-SCID mice coinjected i.v. with [<sup>111</sup>In]In-DOTA-trastuzumab IgG, F(ab')<sub>2</sub> or Fab and [<sup>225</sup>Ac]Ac-DOTA-trastuzumab IgG, F(ab')<sub>2</sub> or Fab.\nRadiation\nabsorbed doses in the tumor and normal organs for [<sup>225</sup>Ac]Ac-DOTA-trastuzumab\nIgG, F(ab')<sub>2</sub> or Fab were estimated based on the biodistribution\nof the [<sup>111</sup>In]In-DOTA-trastuzumab IgG, F(ab')<sub>2</sub> or Fab. Normal tissue toxicity was assessed by hematology\nand blood\nbiochemistry analyses and monitoring body weight in NRG mice injected\ni.v. with 2 and 4 kBq of [<sup>225</sup>Ac]Ac-DOTA-trastuzumab IgG,\nF(ab')<sub>2</sub> or Fab separated by 8 d. RIT studies were\nperformed\nin NRG mice with s.c. 164/8-1B/H2N.luc<sup>+</sup> tumors injected\ni.v. with 2 kBq and 4 kBq of [<sup>225</sup>Ac]Ac-DOTA-trastuzumab\nIgG, F(ab')<sub>2</sub> or Fab separated by 8 d or irrelevant\n[<sup>225</sup>Ac]Ac-DOTA-IgG<sub>1</sub>, two doses of unlabeled\ntrastuzumab\nIgG or 0.9% NaCl. A tumor growth index (TGI) was plotted vs time (d)\nand Kaplan–Meier median survival estimated. [<sup>111</sup>In]In-DOTA-trastuzumab IgG or F(ab')<sub>2</sub> exhibited 4.1-fold\nand 3.3-fold significantly greater tumor uptake at 2 d postinjection\n(p.i.) than Fab at 24 h p.i. However, spleen uptake at 2 d p.i. for\n[<sup>111</sup>In]In-DOTA-trastuzumab IgG was 3.3-fold significantly\nhigher than F(ab')<sub>2</sub> and 13.2-fold higher than Fab\nat 24\nh p.i. [<sup>111</sup>In]In-DOTA-trastuzumab F(ab')<sub>2</sub> and\nFab exhibited higher kidney uptake than IgG. Tumors were imaged by\nSPECT/CT with [<sup>111</sup>In]In-DOTA-trastuzumab IgG and F(ab')<sub>2</sub> but were not well-visualized with [<sup>111</sup>In]In-DOTA-trastuzumab\nFab. The absorbed dose in the tumor was 2.2-fold greater for [<sup>225</sup>Ac]Ac-DOTA-trastuzumab F(ab')<sub>2</sub> than IgG\nand 3.4-fold\ngreater than Fab. Hematological toxicity was observed for [<sup>225</sup>Ac]Ac-DOTA-trastuzumab IgG but not for [<sup>225</sup>Ac]Ac-DOTA-trastuzumab\nF(ab')<sub>2</sub> or Fab. No kidney or liver toxicity or decreased\nbody weight was observed for any RIT agent. Tumor growth was significantly\ninhibited by [<sup>225</sup>Ac]Ac-DOTA-trastuzumab IgG, F(ab')<sub>2</sub> or Fab but [<sup>225</sup>Ac]Ac-DOTA-trastuzumab F(ab')<sub>2</sub> was most effective for increasing median survival (46 d vs\n22 d for IgG and 29 d for Fab). We conclude that [<sup>111</sup>In]In-\nand [<sup>225</sup>Ac]Ac-DOTA-trastuzumab F(ab')<sub>2</sub> exhibited\nsuperior properties for theranostic imaging and α-particle RIT\nof HER2-positive human BC xenografts in NRG mice.