6zvf
Crystal structure of the recombinant Fab fragment derived from the hybridoma M3/38 in complex with a human Galectin-3 peptide
Structural highlights
FunctionLEG3_HUMAN Galactose-specific lectin which binds IgE. May mediate with the alpha-3, beta-1 integrin the stimulation by CSPG4 of endothelial cells migration. Together with DMBT1, required for terminal differentiation of columnar epithelial cells during early embryogenesis (By similarity). In the nucleus: acts as a pre-mRNA splicing factor. Involved in acute inflammatory responses including neutrophil activation and adhesion, chemoattraction of monocytes macrophages, opsonization of apoptotic neutrophils, and activation of mast cells.[1] [2] [3] Publication Abstract from PubMedThe 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 alphahGal3 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 V(H)/V(L) 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 (K(D) = 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 (89)Zr(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 alphahGal3 Fab offers clinical potential for the diagnostic imaging of TC. Effective rational humanization of a PASylated anti-galectin-3 Fab for the sensitive PET imaging of thyroid cancer in vivo.,Peplau E, De Rose F, Eichinger A, Reder S, Mittelhauser M, Scafetta G, Schwaiger M, Weber WA, Bartolazzi A, D'Alessandria C, Skerra A Sci Rep. 2021 Apr 1;11(1):7358. doi: 10.1038/s41598-021-86641-0. PMID:33795750[4] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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