Evaluation of Cu-64-anti-RAGE scFv for assessment of pancreatic cancer-associated RAGE expression

Objectives Noninvasive detection of early pancreatic cancer could improve patient survival, as this disease is notorious for poor prognosis. Despite extensive efforts, there are very few specific biomarkers available for diagnosis or targeted therapy in pancreatic ductal adenocarcinoma (PDAC). Over-expression of the receptor for advanced glycation end products (RAGE) has been identified as a novel biomarker for neoplastic lesions and PDAC. In this study, a RAGE-specific antibody fragment (scFv) was developed as a specific imaging agent for pancreatic cancer with PET.

Methods An anti-RAGE scFv was expressed from a humanized anti-RAGE mAb (3B4) sequence. A non-binding scFv, M4, was prepared by grafting the complementarity determining regions of 3B4 to a different framework. Both scFvs were functionalized with sulfo-Cy5 and a radioisotope metal chelator (NOTA). Cu-64-NOTA-3B4 and Cu-64-NOTA-M4 were administered in a syngeneic mouse model of pancreatic cancer (Panc02) to evaluate receptor mediated tumor uptake relative to normal tissues.

Results Surface plasmon resonance analysis demonstrated high affinity for 3B4 and its conjugates and also demonstrated that M4 did not bind RAGE. Immunofluorescence microscopy demonstrated that 3B4 bound RAGE expressed on mouse pancreatic adenocarcinoma Panc02. In tissues known to be enriched in RAGE, which include liver, kidney, and spleen, there were statistically significant differences in uptake of Cu-64-NOTA-3B4 relative to the non-binding control Cu-64-NOTA-M4. However, there were no significant differences in tumor uptake.

Conclusions Due to rapid blood clearance typical of scFvs, tumor uptake of Cu-64-NOTA-3B4 was relatively low but resulted in statistically significant tumor/blood ratios relative to the non-binding control (3B4: 1.65 ± 0.45, M4: 0.64 ± 0.15, p < 0.05, unpaired t-test). Cu-64-NOTA-3B4 demonstrated apparent RAGE-mediated uptake in tissues that are known to be enriched in RAGE, thus demonstrating that this scFv is functional. Future work will include the development of slower clearing antibody fragments and overcoming uptake in tissues that serve as an antigen sink.