Abstract Despite clear advantages of brachytherapy over external beam radiotherapy, current implementations of this approach have several limitations. These include the need for general anesthesia or intravenous sedation, complicated placement procedures and the need for post -treatment re-excision for device removal. An alternative approach that circumvents these constraints would be clearly attractive. In a previous study, we demonstrated that a thermally sensitive elastin-like polypeptide (ELP) displayed increased tumor retention through a thermally triggered in-situ coacervation and delayed tumor growth through the therapeutic action of a conjugated radionuclide, 131I, over a non-thermally sensitive ELP following intratumoral (i.t.) administration. However, this “smart” radionuclide carrier did not lead to complete tumor regression and had a small effect on survival. To improve the antitumor efficacy of this thermally sensitive ELP-radionuclide conjugate (ELP-depot), we investigated the effects of three orthogonal parameters upon radionuclide retention in tumor following i.t administration: the ELP concentration, molecular weight and tyrosine content (hydrophobicity). Furthermore, using the ELP-depot optimized in the above studies, we examined the antitumor efficacy and toxicity in two human tumors (human head and neck squamous carcinoma (FaDu) and human prostate (PC-3) tumor) xenografts in nude mice. The results demonstrate that the injected concentration, the transition temperature (Tt), and the hydrophobicity are critical design parameters with significant influence over radionuclide tumor retention. A 16-fold increase in concentration from 62.5 μM to 1000 μM resulted in a 5-fold increase in retention (16% ID versus 85% ID) 7 days post-injection (p < 0.01, ANOVA). Decreasing the Tt (a value dependent upon the molecular weight) from 30 °C to 24 °C resulted in a 14-fold increase in tumor retention over the same time period (p < 0.01, ANOVA). Tyrosine content-induced the changes of ELP hydrophobicity have significantly affected the behavior of radionuclide in tumor, presenting that the tumor retention, radioactivity stability and antitumor efficacy were enhanced with the increase in the tyrosine number from 1,4 to 7. This optimized ELP depot achieved a 100% of tumor response to this radiotherapy in both tumor xenografts and no significant toxicity was found in either of tumor models. Interestingly, complete tumor regression (tumor size turned out to be zero at the end of the experiment) rate was 67% in FaDu and 77% in PC-3. Together, all our data indicates that the ELP radionuclide depot is an ideal intratumoral radionuclide carrier, because it is injectable, biocompatible, has long tumor retention and good antitumor efficacy for local radiotherapy of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2884. doi:1538-7445.AM2012-2884