Development of a chimeric Fab directed against human galectin-3 and validation as an immune-PET tracer for the sensitive in vivo imaging of thyroid cancer

BACKGROUND The lack of facile methods for the specific characterization of malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Due to its restricted expression in such nodules, the cell-associated lectin Galectin-3 (Gal3) has emerged as a marker for TC with growing interest for in vivo imaging as well as targeted radionuclide therapy. To accelerate translation into clinical application, we have developed a cognate chimeric human Fab fragment derived from the rat anti-Gal3 monoclonal antibody (mAb) M3/38. METHODS The variable immunoglobulin (Ig) light and heavy chain sequences were cloned from a hybridoma cell line, and the corresponding Fab fragment carrying human IgG1/ constant genes was functionally produced in the periplasm of E. coli and purified to homogeneity. To moderately prolong its plasma half-life and, thus, increase tumor uptake, the recombinant Fab was fused with a long disordered amino acid chain comprising Pro, Ala and Ser residues (PASylation). This novel tracer was subjected to in vitro characterization and in vivo validation using two thyroid orthotopic murine models. To this end, the αGal3-Fab-PAS200 was conjugated with Dfo, labeled with 89Zr under mild conditions and tested for binding on TC cell lines. Athymic nude mice were inoculated either with FRO82-1 or with CAL62 tumor cells into the left thyroid lobe. After intravenous injection with ~3.0 MBq of 89Zr-DFO-PAS200-Fab, these mice were subjected to PET/CT imaging followed by quantification of tumor accumulation and immunohistochemical analysis. RESULTS The αGal3-Fab-PAS200 revealed high affinity towards the recombinant Gal3 antigen, with a dissociation constant ≤1 nM as measured via ELISA, surface plasmon resonance spectroscopy and radioactive cell binding assay. The in vivo Gal3-targeting by the 89Zr(IV)-labeled protein tracer, as investigated by immuno-PET, demonstrated highly selective and fast accumulation in orthotopically implanted tumors, with strong contrast images achieved 24 h post-injection, and no uptake in the tumor-free thyroid lobe, as also confirmed by biodistribution studies. CONCLUSIONS The chimeric αGal3 89Zr-DFO-PAS200-Fab tracer has selective accumulation in the tumor-bearing thyroid lobe of xenograft mice. Thus, this novel radioactive probe offers potential to change thyroid cancer management, in addition to current, and to reduce unnecessary thyroidectomies.