Bispecific T-cell engager (BiTE) molecules exert antitumor activity by binding one arm to CD3 on cytotoxic T cells and the other arm to a tumor-associated antigen. Methods: We generated a fully mouse cross-reactive mesothelin-targeted BiTE molecule that is genetically fused to an Fc-domain for half-life extension, and we evaluated the biodistribution and tumor targeting of a ⁸⁹Zr-labeled mesothelin half-life–extended (HLE) molecule in 4T1 breast cancer–bearing syngeneic mice with PET. The biodistribution of 50 μg of ⁸⁹Zr-mesothelin HLE BiTE was studied over time by PET imaging in BALB/c mice and revealed uptake in tumor and lymphoid tissues with an elimination half-life of 63.4 h. Results: Compared with a nontargeting ⁸⁹Zr-control HLE BiTE, the ⁸⁹Zr-mesothelin HLE BiTE showed a 2-fold higher tumor uptake and higher uptake in lymphoid tissues. Uptake in the tumor colocalized with mesothelin expression, whereas uptake in the spleen colocalized with CD3 expression. Evaluation of the effect of protein doses on the biodistribution and tumor targeting of ⁸⁹Zr-mesothelin HLE BiTE revealed for all dose groups that uptake in the spleen was faster than in the tumor (day 1 vs. day 5). The lowest dose, 10 μg, of ⁸⁹Zr-mesothelin HLE BiTE had higher spleen uptake and faster blood clearance than the higher doses, 50 and 200 μg. ⁸⁹Zr-mesothelin HLE BiTE tumor uptake was similar at all doses. Conclusion: The mesothelin HLE BiTE showed specific tumor uptake, and both arms contributed to the biodistribution profile. These findings support the potential for clinical translation of HLE BiTE molecules.

双特异性 T 细胞接合器 (BiTE) 分子通过将一个臂与细胞毒性 T 细胞上的 CD3 结合，将另一臂与肿瘤相关抗原结合来发挥抗肿瘤活性。方法：我们生成了一种完全小鼠交叉反应的间皮素靶向 BiTE 分子，该分子通过基因融合到 Fc 结构域以延长半衰期，并且我们评估了⁸⁹ Zr 标记的半衰期延长的间皮素的生物分布和肿瘤靶向性。 (HLE) 分子在 4T1 乳腺癌同基因小鼠中通过 PET 检测。通过 PET 成像在 BALB/c 小鼠中研究了 50 μg ⁸⁹ Zr-间皮素 HLE BiTE 随着时间的推移的生物分布，结果显示肿瘤和淋巴组织中的吸收，消除半衰期为 63.4 小时。结果：与非靶向⁸⁹ Zr 对照 HLE BiTE 相比， ⁸⁹ Zr-间皮素 HLE BiTE 的肿瘤摄取量高出 2 倍，淋巴组织摄取量也更高。肿瘤中的摄取与间皮素表达共定位，而脾中的摄取与CD3表达共定位。蛋白质剂量对⁸⁹ Zr-间皮素 HLE BiTE 的生物分布和肿瘤靶向影响的评估显示，对于所有剂量组，脾脏中的摄取速度快于肿瘤中的摄取速度（第 1 天与第 5 天）。最低剂量（10 μg）的⁸⁹ Zr-间皮素 HLE BiTE 比较高剂量（50 和 200 μg）具有更高的脾脏摄取和更快的血液清除率。 ⁸⁹ Zr-间皮素 HLE BiTE 肿瘤摄取在所有剂量下均相似。结论：间皮素 HLE BiTE 显示出特异性肿瘤摄取，并且双臂均有助于生物分布。这些发现支持 HLE BiTE 分子临床转化的潜力。