Development of an ¹⁸F-labeled anti-human CD8 VHH for same-day immunoPET imaging

Purpose: Cancer immunotherapies (CITs) have revolutionized the treatment of certain cancers, but many patients fail to respond or relapse from current therapies, prompting the need for new CIT agents. CD8⁺ T cells play a central role in the activity of many CITs, and thus, the rapid imaging of CD8⁺ cells could provide a critical biomarker for new CIT agents. However, existing ⁸⁹Zr-labeled CD8 PET imaging reagents exhibit a long circulatory half-life and high radiation burden that limit potential applications such as same-day and longitudinal imaging.

Methods: To this end, we discovered and developed a 13-kDa single-domain antibody (VHH5v2) against human CD8 to enable high-quality, same-day imaging with a reduced radiation burden. To enable sensitive and rapid imaging, we employed a site-specific conjugation strategy to introduce an ¹⁸F radiolabel to the VHH.

Results: The anti-CD8 VHH, VHH5v2, demonstrated binding to a membrane distal epitope of human CD8 with a binding affinity (K_D) of 500 pM. Subsequent imaging experiments in several xenografts that express varying levels of CD8 demonstrated rapid tumor uptake and fast clearance from the blood. High-quality images were obtained within 1 h post-injection and could quantitatively differentiate the tumor models based on CD8 expression level.

Conclusion: Our work reveals the potential of this anti-human CD8 VHH [¹⁸F]F-VHH5v2 to enable rapid and specific imaging of CD8⁺ cells in the clinic.