A FABulous approach to image human macrophages

Imaging tumor-associated macrophages (TAMs) may reflect prognosis, therapeutic response and radiotheranostic potential. We report the discovery and development of a synthetic human Fab as the first immunoPET tracer targeting human CD68 (huCD68), the gold standard histopathological biomarker of pan-macrophages.

Phage display was used to identify novel huCD68-specific Fabs. The lead Fab for immunoPET development was chosen based on expression yield and stability. Fab was conjugated to p-SCN-Bn-DFO and radiolabeled with ⁸⁹Zr, yielding [⁸⁹Zr]Zr-DFO-Fab. Stability and specific binding of the tracer to THP-1 macrophages were assessed in vitro. The specificity of the tracer in vivo was characterized by PET imaging of two different animal models: 1) BALB/c mice bearing huCD68 antigen- or irrelevant protein-plugs, and 2) tumor xenografted NSG mice intratumorally injected with either THP-1 macrophages or THP-1 undifferentiated cells. Finally, tumors were subjected to immunofluorescence (IF) staining of huCD68 and correlated with PET standardized uptake values (SUV).

Four novel huCD68-specific Fabs were identified. Fab8 was chosen as the lead based on high expression yield, purity, and stability. In vivo, [⁸⁹Zr]Zr-DFO-Fab8 uptake was significantly higher in huCD68 antigen-plugs than in irrelevant protein-plugs at 1- and 4-h post-injection (p.i.). Tracer uptake in THP-1 macrophage tumors was also significantly higher than in THP-1 undifferentiated tumors at 1- and 6-h p.i.. IF of tumors confirmed higher density of huCD68 in those implanted with THP-1 macrophages than those with THP-1 undifferentiated cells. CD68 expression highly correlated with immunoPET SUV.

[⁸⁹Zr]Zr-DFO-Fab8 binds specifically to huCD68, supporting the feasibility of direct PET-to-histopathogical correlation of total TAM burden.