Tissue factor-specific sdAb radiotracers for molecular imaging in solid tumors

Tissue factor (TF) expression has been associated with tumor aggressiveness and poor prognosis in several cancers. With the recent FDA approval of an antibody-drug-conjugate targeting TF, tisotumab vedotin for recurrent metastatic cervical cancer, companion diagnostic tools capable of capturing tumor heterogeneity are increasingly critical. In this study, we developed and characterized single domain antibody (sdAb)-based radiotracers to non-invasively visualize tumors expressing either murine (m) or human (h) TF protein. SdAbs targeting hTF and mTF were obtained from an immune llama library. Seven candidates targeting each species’ orthologue were selected, characterized in vitro and radiolabeled with 99mTc for in vivo screening in athymic nude mice bearing both mTF and hTF positive tumors. Lead anti-hTF sdAb was radiolabeled with 68Ga for PET imaging. In vitro, all sdAbs exhibited excellent affinities for their respective targets, reaching the picomolar range. When radiolabeled with 99mTc, the lead compounds showed strong uptake and specific binding in TF-positive tumors as confirmed by SPECT imaging. The lead sdAb 64B4 targeting hTF protein, demonstrated an uptake of 8.07±1.02% IA/g in BxPC-3 tumors and a tumor-to-blood ratio of 26.06±4.36. The best candidate for targeting mTF protein, sdAb 66A12, displayed an uptake of 3.30±0.22% IA/g in mTF positive tumors and a tumor-to-blood ratio of 10.70±2.83. Off-tumor uptake was detected in several organs, with unexpectedly the highest levels detected in brown adipose tissue, consistent with mTF protein expression levels determined by ELISA. We successfully developed and validated sdAb-based radiotracers targeting either mTF or hTF, enabling the noninvasive visualization of TF-expressing tumors.