4.8.4. Evaluation of [68Ga]**3** in Mice Pre-Treated with **4**

Female BALB/c mice were administered i.v. with **4** (100 μg, 0.25 μmol) in saline (100 μL) (n = 4). Four mice without pre-treatment were included as controls. After 1 h [68Ga]**3** (mean 11 MBq, 8–13 μg, 6.4–10 nmol) in < 10% EtOH/PBS (100 μL) was administered i.v. PET/CT images were obtained 2 p.i. Following the scan, the mice were euthanized, and the knee, shoulder, and muscle tissue were collected and weighed. Sample radioactivity was measured using a gamma counter and the %ID/g values were calculated after correcting for decay and injected amount of radioactivity.

#### **5. Conclusions**

Within this study, we demonstrated that [68Ga]**3** can serve as a PET alternative for pretargeted imaging, even though it displayed lower accumulation values than the 'Gold-standard' for pretargeted imaging, [111In]**2.**

**Supplementary Materials:** The following are available online. Analytical HPLC data of [111In]2 and [68Ga]3 (Figures S1 and S2), RadioTLC data of [68Ga]3 (Figure S3), additional PET biodistribution data (Table S1) and SPECT biodistribution data (Table S2) are reported.

**Author Contributions:** Conceptualization, P.E.E., M.M.H., and A.K.; Synthesis, P.E.E., A.Y., J.F.V., R.R., and M.R.; Animal experiments, J.T.J. and K.N.; Writing—Original draft preparation, P.E.E., J.T.J. and K.N.; Writing—Review and editing, M.M.H. and A.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project received funding from the European Union's Horizon 2020 research and innovation programme under grant agreements no. 668532 (Click-It) and 670261 (ERC Advanced Grant), the Lundbeck Foundation, the Novo Nordisk Foundation, the Innovation Fund Denmark, the Danish Cancer Society, Arvid Nilsson Foundation, Svend Andersen Foundation, the Neye Foundation, the Research Foundation of Rigshospitalet, the Danish National Research Foundation (grant 126), the Research Council of the Capital Region of Denmark, the Danish Health Authority, the Carlsberg Foundation, the John and Birthe Meyer Foundation and the Research Council for Independent Research.

**Acknowledgments:** This project has received funding from the European Union's EU Framework Programme for Research and Innovation Horizon 2020, under grant agreement no. 668532., from the European Union's EU Framework Programme for Research and Innovation Horizon 2020 (grant agreement no. 670261), the Lundbeck Foundation, the Novo Nordisk Foundation, the Innovation Fund Denmark, and the Research Council for Independent Research.

**Conflicts of Interest:** No conflict of interest has to be reported.
