5cma

Publication Abstract from PubMed

B7-H3 (CD276) is both an inhibitory ligand for natural killer cells and T cells, and a tumor antigen that is widely expressed among human solid tumors. Anti-B7-H3 mouse monoclonal antibody 8H9 has been successfully used for radioimmunotherapy for patients with B7-H3(+) tumors. We present the humanization, affinity maturation and epitope mapping of 8H9 based on structure determination, modeling and yeast display methods. The crystal structure of ch8H9 Fab fragment was solved to 2.5 A resolution and used as a template for humanization. By displaying the humanized 8H9 single chain Fv (ScFv) on the surface of yeast, the affinity was matured by sequential random mutagenesis and fluorescent cell sorting. Six mutations (three in the CDR and three in the framework regions) were identified and incorporated into an affinity-matured humanized 8H9 construct (hu8H9-6m) and an affinity-matured chimeric 8H9 construct (ch8H9-6m). The hu8H9-6m scFv had a 160-fold improvement in affinity (0.9 nM KD) compared to parental hu8H9 scFv (144 nM KD). The IgG formats of ch8H9-6m and hu8H9-6m (nanomolar to sub-nanomolar KD) had 2- to 9-fold enhancements in affinity compared to their parental forms, potent in vitro antibody dependent cell-mediated cytotoxicity (0.1-0.3 mug/mL EC50), and high tumor uptake in mouse xenografts. Based on in silico docking studies and experimental validation, the precise molecular epitope of 8H9 was determined to be the FG loop of B7-H3, a region critical to its function in immunologic blockade, unique among anti-B7-H3 antibodies published to date.

Humanized Affinity-Matured Monoclonal Antibody 8H9 Has Potent Anti-Tumor Activity and Binds to FG Loop of B7-H3.,Ahmed M, Cheng M, Zhao Q, Goldgur Y, Cheal SM, Guo HF, Larson SM, Cheung NV J Biol Chem. 2015 Oct 20. pii: jbc.M115.679852. PMID:26487718^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.