<p>STA551 is an anti-human CD137 (hCD137) switch antibody with adenosine triphosphate (ATP)-dependent antigen binding, developed to achieve potent anti-tumor effects and superior safety. Based on several reports, unlike in normal tissues, high concentrations of ATP are considered to exist in the interstitial space of tumors, thus STA551 is expected to exhibit tumor-selective antigen (hCD137) binding (ATP switch concept). This study aims to quantitatively investigate this ATP switch concept <i>in vivo</i> by combining tissue distribution studies and physiologically based pharmacokinetic (PBPK) modeling. An iodinated conventional anti-hCD137 antibody, ¹²⁵I-Ure-mIgG1, was administered as a tracer to tumor-bearing hCD137 knock-in (KI) mice. Co-administration of excess unlabeled Ure-mIgG1 at 20&#xa0;mg/kg increased plasma tracer radioactivity levels and decreased the tissue-to-plasma (T/P) ratios in several normal tissues and tumors. However, co-administration of unlabeled Sta-MB (mouse surrogate antibody of STA551) at 1 or 20&#xa0;mg/kg did not change the plasma tracer radioactivity levels but clearly reduced T/P ratios, mainly in tumors, strongly suggesting tumor-selective binding of Sta-MB. A PBPK model was developed to explain this distribution data, and parameters including non-specific clearance, interstitial uptake clearance, target-related parameters, and ATP switch molecule concentrations were estimated. Importantly, the ATP switch molecule concentration in the tumor interstitial space was estimated to reach approximately hundreds μM, with much lower concentrations in non-tumor tissues. In conclusion, this combination approach successfully demonstrated the ATP switch concept of STA551 <i>in vivo</i>. These findings will help guide clinical trials for STA551 and the development of future switch antibodies.</p> Graphical Abstract <p></p>