Zirconium-89 Labeling and Preliminary Evaluation of Simtuzumab as a Candidate Positron Emission Tomography (PET) Imaging Agent for Human Lysyl Oxidase-like 2 (LOXL2) in the Extracellular Matrix of Solid Tumors

Abstract Lysyl oxidase-like 2 (LOXL2) is a copper-dependent enzyme that is involved in collagen and elastin cross-linking in the tumor extracellular matrix (ECM) and contributes to tumor progression. Simtuzumab is a clinically evaluated humanized monoclonal antibody that binds to an allosteric binding site on LOXL2. Herein, we report radiolabeling and preliminary evaluation of simtuzumab labeled with zirconium-89 (89Zr, t1/2: 3.3 days) for positron emission tomography (PET) imaging of LOXL2 in the ECM of tumors. Simtuzumab was conjugated with desferrioxamine (DFO) and labeled with 89Zr in >98% yield. Magnetic bead-based assay confirmed specific binding of [89Zr]Zr-DFO-simtuzumab to LOXL2. LOXL2 expression levels and cell binding of [89Zr]Zr-DFO-simtuzumab were assessed in MIA PaCa-2 and HepG2 cell lines. Western blot analysis indicated higher LOXL2 expression in MIA PaCa-2 cells compared with HepG2 cells. Cell binding assays revealed minimal binding (<0.5%) of [89Zr]Zr-DFO-simtuzumab in both cell lines, reflecting the lack of cell surface expression of LOXL2. Immunohistochemical (IHC) studies revealed primarily intracellular localization of LOXL2 in the MIA PaCa-2 xenograft model, with minimal expression in HepG2 xenografts in mice. microPET/CT imaging studies in mice bearing MIA PaCa-2 xenografts demonstrated low but visible uptake of [89Zr]Zr-DFO-simtuzumab in tumors (6.2 ± 1.3% ID/g) at 96 h postinjection. Ex vivo biodistribution studies indicated higher uptake of the labeled conjugate in both xenograft tumor models relative to muscle, but no significant differences were observed in [89Zr]Zr-DFO-simtuzumab uptake in the two tumor models, consistent with the low ECM-associated LOXL2 in IHC staining. Overall, simtuzumab has been successfully labeled with [89Zr]Zr using DFO and characterized as a candidate immunoPET tracer for LOXL2. However, additional studies are needed to further validate the labeled conjugate in tumor models with high LOXL2 expression in the ECM and to confirm LOXL2-specific ECM imaging in vivo by PET.