Development and characterization of a fully human antibody targeting SCF/c-kit signaling

CD117/c-kit, a tyrosine kinase receptor, plays a critical role in hematopoiesis, pigmentation, and fertility. The overexpression and activation of c-kit are thought to promote tumor growth and have been reported in various cancers, including leukemia, glioblastoma and mastocytosis. To disrupt the SCF/c-kit signaling axis in cancer, we generated a c-kit antagonist human antibody (NN2101) that binds to domain 2/3 of c-kit. This completely blocked the SCF-mediated phosphorylation of c-kit and inhibited TF-1 cell proliferation, erythroleukemia. In ad-dition, the examination of binding affinity using surface plasmon resonance (SPR) assay showed that NN2101 can bind to c-kit of monkeys (K-D = 2.92 x 10(-10) M), rats (K-D = 1.68 x 10(-6) M), mice (K-D = 11.5 x 10(-9) M), and humans (KD = 2.83 x 10(-12) M). We showed that NN2101 does not cause antibody-dependent cell-mediated cy-totoxicity and complement-dependent cytotoxicity. The immunogenicity of NN2101 was similar to that of bevacizumab. Furthermore, the crystal structure of NN2101 Fab was determined and the structure of NN2101 Fab:c-kit complex was modeled. Structural information, as well as mutagenesis results, revealed that NN2101 can bind to the SCF-binding regions of c-kit. Collectively, we generated a c-kit neutralizing human antibody (NN2101) for the treatment of erythroleukemia and characterized its biophysical properties. NN2101 can poten-tially be used as a therapeutic antibody to treat different cancers. (C) 2020 Published by Elsevier B.V.