Purpose <p>AG10 is a molecule that can bind to the thyroxine (T4) binding pocket of transthyretin (TTR). The reversible association between the hydrophilic peptides and TTR using AG10 could increase the circulation half-life (t_1/2). We hypothesized that modifying PSMA-617 with AG10 could extend the t_1/2 of PSMA-617 and mitigate the limitations caused by insufficient uptake. The aim of this study was to develop an optimal AG10-modified radiotherapeutic agent that could maximize both tumor uptake and absorbed dose, thereby enhancing therapeutic efficacy for the treatment of tumors, even with moderate PSMA expression.</p> Methods <p>Lysine was used as a linker for the conjugation of PSMA-617 and AG10 to synthesize AG10-PSMA-617 (AP-01). AP-01 was labeled with ⁶⁸Ga to image prostate carcinoma xenografts with moderate PSMA expression, serving as a reference for PSMA radio-ligand therapy (PRLT) based treatments. AP-01 was labeled with ¹⁷⁷Lu for the treatment of prostate carcinoma xenografts with moderate PSMA expression. The binding properties and biodistribution of ⁶⁸Ga-AP-01 and ¹⁷⁷Lu-AP-01 were studied both in vitro and in vivo. The cytotoxic effects of the ¹⁷⁷Lu-AP-01 were evaluated in prostate carcinoma xenografts.</p> Results <p>PSMA-617 was successfully conjugated with AG10 using lysine as the linker and labeled with ¹⁷⁷Lu to produce PRLT radiopharmaceuticals-¹⁷⁷Lu-AP-01. AP-01 was labeled with ⁶⁸Ga for imaging prostate carcinoma with moderate PSMA expression. AP-01 demonstrated a high binding affinity (IC₅₀ = 3.42 nM) for PSMA in vitro, comparable to that of PSMA-617 (IC₅₀ = 3.38 nM) (<i>P</i> = 0.324). PET/CT imaging of ⁶⁸Ga-AP-01 demonstrated significantly improved tumor uptake and retention as compared with ⁶⁸Ga-PSMA-617. The maximum uptake of ⁶⁸Ga-AP-01 and ⁶⁸Ga-PSMA-617 by 22Rv1 tumors was 18.07 ± 1.19%ID/g and 9.27 ± 1.20%ID/g at 4&#xa0;h pi, respectively (<i>P</i> &lt; 0.05). The in vivo targeting abilities of ¹⁷⁷Lu-AP-01 were evaluated through SPECT/CT imaging. The tumor (maximum)-to-muscle (maximum) (T/Mmax) ratio of ¹⁷⁷Lu-AP-01 was 49.28 ± 7.18 at 72&#xa0;h pi. However, the T/Mmax ratio of ¹⁷⁷Lu-PSMA-617 was 8.39 ± 8.57 at 48&#xa0;h pi. Biodistribution studies further confirmed the significantly higher tumor uptake of ¹⁷⁷Lu-AP-01 (4.87 ± 0.91%ID/g) compared to ¹⁷⁷Lu-PSMA-617 (1.12 ± 0.01%ID/g) at 96&#xa0;h post-injection (<i>P</i> &lt; 0.05). The inhibitory efficiency of ¹⁷⁷Lu-AP-01 on 22Rv1 tumor xenografts was 88.36%, which was superior to that of ¹⁷⁷Lu-PSMA-617 (73.36%) (<i>P</i> &lt; 0.05). ¹⁷⁷Lu-AP-01 demonstrated no toxic effects on hepatorenal function, routine blood tests, or major organs in mice when compared to the control group.</p> Conclusion <p>In this study, AP-01 was successfully synthesized. ⁶⁸Ga-AP-01 and ¹⁷⁷Lu-AP-01 were obtained with high radiochemical purity and stability. High binding affinity and PSMA targeting specificity were identified in vitro and in vivo. With significantly enhanced tumor uptake and retention, ¹⁷⁷Lu-AP-01 has the potential to improve therapeutic efficacy for the treatment of prostate cancer with moderate PSMA expression.</p>