Longitudinal Imaging Reveals Tumor Uptake and Prolonged Retention of Bi-Specific T-Cell Engaging Antibody in GBM via Passive and Active Mechanisms

Abstract Purpose: This study aimed to assess the dynamics of tumor-specific uptake, retention, and blood-tumor barrier (BTB) penetration of our unique IL13Rα2xCD3 bispecific T-cell engager (BiTE) following systemic administration in mice with intracranial GBM xenografts. Experimental Design: In vitro, BiTE binding and accumulation were evaluated in glioma neurospheres. In in vivo studies, the BiTE labeled with either 124I using residualizing chemistry or conjugated to Cy-5 were used for longitudinal tracking in patient-derived (PDX) GBM models using PET/CT and confocal microscopy. The survival analysis in mice bearing intracranial GBM tumors was conducted to validate the findings from imaging studies. Results: In vitro, the BiTE demonstrated target-specific binding and accumulation in IL13Rα2-expressing glioma spheres. In vivo, PET/CT imaging revealed that the BiTE reached the tumor site within 3h post-injection, achieving up to 4.8% ID/g, with sustained tumor retention for up to 24 h, significantly higher than background levels in surrounding normal brain tissue. Confocal microscopy confirmed BiTE presence in the tumor bed extravascular space with evidence of T-cell-mediated BiTE transport across the BTB. Despite its short plasma half-life, the BiTE remained in the tumor microenvironment for at least 24h. Mice bearing GBM6 brain tumors treated with BiTE for 3-4 days apart via i.v. route showed a significant survival advantage over the control group. Conclusion: Our findings provide critical insights into the pharmacokinetics of BiTE molecules in GBM. They demonstrate effective penetration and prolonged intratumoral retention following a single systemic dose, supporting further exploring BiTE treatment regimens for translation to clinical settings.