PET of CD105 expression with a ^61/64Cu-labeled Fab antibody fragment

Objectives To generate and characterize the Fab fragment of TRC105, a monoclonal antibody that binds with high affinity to human and murine CD105 (i.e. endoglin), and investigate its potential for positron emission tomography (PET) imaging of tumor angiogenesis in a mouse model after ^61/64Cu-labeling.

Methods TRC105-Fab was generated by enzymatic papain digestion. The integrity and CD105 binding affinity of TRC105-Fab was evaluated before NOTA (i.e., 1,4,7-triazacyclononane-1,4,7-triacetic acid) conjugation and ^61/64Cu-labeling. Serial PET imaging and biodistribution studies were carried out in the 4T1 murine breast cancer model to quantify tumor targeting and normal organ distribution of ^61/64Cu-NOTA-TRC105-Fab. Blocking studies with TRC105, as well as histological assessment, were performed to confirm CD105 specificity of the tracer.

Results TRC105-Fab was produced with high purity through papain digestion of TRC105, as confirmed by SDS-PAGE, HPLC analysis, and mass spectrometry. ^61/64Cu-labeling of NOTA-TRC105-Fab was achieved with good yield and specific activity. PET imaging revealed rapid uptake of ⁶⁴Cu-NOTA-TRC105-Fab in the 4T1 tumor (3.6±0.4, 4.2±0.5, 4.9±0.3, and 4.6±0.8 %ID/g at 0.5, 2, 5, and 24 h post-injection respectively; n = 4). Since tumor uptake peaked soon after tracer injection, ⁶¹Cu-labeled TRC105-Fab was also able to provide good tumor contrast at 3 and 8 h post-injection. CD105 specificity of the tracer was confirmed with blocking and histology studies.

Conclusions ^61/64Cu-NOTA-TRC105-Fab exhibited prominent and target specific uptake in the 4T1 tumor, as seen with PET. The use of a Fab fragment led to much faster tumor uptake (peak uptake was noted a few hours after injection) compared to radiolabeled intact antibody, which may be translated into same day immunoPET imaging for clinical investigation. The use of ⁶¹Cu, which has higher β⁺ branching ratio than ⁶⁴Cu (62% vs 17%), is optimal for small proteins like Fab. It offers stronger signal intensity and requires a lower tracer dose for PET imaging than ⁶⁴Cu, which will facilitate clinical translation.