Effective rational humanization of a PASylated anti-galectin-3 Fab for the sensitive PET imaging of thyroid cancer in vivo

Abstract

The lack of a non-invasive test for malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Human galectin-3 (hGal3) has emerged as a promising target for medical TC imaging and diagnosis because of its exclusive overexpression in malignant thyroid tissues. We previously developed a human-chimeric αhGal3 Fab fragment derived from the rat monoclonal antibody (mAb) M3/38 with optimized clearance characteristics using PASylation technology. Here, we describe the elucidation of the hGal3 epitope recognized by mAb M3/38, X-ray crystallographic analysis of its complex with the chimeric Fab and, based on the three-dimensional structure, the rational humanization of the Fab by CDR grafting. Four CDR-grafted versions were designed using structurally most closely related fully human immunoglobulin VH/VL regions of which one—employing the acceptor framework regions of the HIV-1 neutralizing human antibody m66—showed the highest antigen affinity. By introducing two additional back-mutations to the rodent donor sequence, an affinity toward hGal3 indistinguishable from the chimeric Fab was achieved (KD = 0.34 ± 0.02 nM in SPR). The PASylated humanized Fab was site-specifically labelled with the fluorescent dye Cy7 and applied for the immuno-histochemical staining of human tissue sections representative for different TCs. The same protein was conjugated with the metal chelator Dfo, followed by radiolabelling with 89Zr(IV). The resulting protein tracer allowed the highly sensitive and specific PET/CT imaging of orthotopic tumors in mice, which was confirmed by quantitative analysis of radiotracer accumulation. Thus, the PASylated humanized αhGal3 Fab offers clinical potential for the diagnostic imaging of TC.

Preclinical Evaluation of 99mTc-MAG3-5-Fab Targeting TREM2 in Lung Cancer Mouse Models: A Comparison with 99mTc-MAG3-5-F(ab′)2

“…To overcome the disadvantage of extended circulation time of the antibody in the blood, two antibody fragments, Fab and F­(ab′) 2 , were prepared via enzymatic digestion. Neither fragment contained the Fc segment, thus reducing the nonspecific binding and the molecular mass. − The molecular masses of Fab and F­(ab′) 2 were approximately 50 and 100 kDa, respectively. The Fab fragment had one antigen-binding domain, while the F­(ab′) 2 fragment had two antigen-binding domains.…”

ImmunoPET: Antibody-Based PET Imaging in Solid Tumors

“…Moreover, β-galactoside-binding protein galectin-3 (Gal-3) is a biomarker, expressed only in thyroid cancer cells (63). 89 Zrlabeled targeting IgG and Fab fragments successfully detected thyroid cancer lesions and can potentially be used for identifying recurrence and metastasis (277)(278)(279)(280)(281).…”

Next-Generation Molecular Imaging of Thyroid Cancer

“…Some Fabs have been discovered for the treatment of TC (targeting cluster of differentiation 276 [CD276] [138], etc. ), and some publications reported the potential value of Fab as diagnostic probes targeting Galectin-3 [125,139,140].…”