NN-2101

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 addition, 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 × 10^-10 M), rats (K_D = 1.68 × 10^-6 M), mice (K_D = 11.5 × 10^-9 M), and humans (K_D = 2.83 × 10^-12 M). We showed that NN2101 does not cause antibody-dependent cell-mediated cytotoxicity 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 potentially be used as a therapeutic antibody to treat different cancers.

Stem cell factor (SCF) and its receptor, cKIT, are novel regulators of pathological neovascularization in the eye, which suggests that inhibition of SCF/cKIT signaling may be a novel pharmacological strategy for treating neovascular age-related macular degeneration (AMD). This study evaluated the therapeutic potential of a newly developed fully human monoclonal antibody targeting cKIT, NN2101, in a murine model of neovascular AMD. In hypoxic human endothelial cells, NN2101 substantially inhibited the SCF-induced increase in angiogenesis and activation of the cKIT signaling pathway. In a murine model of neovascular AMD, intravitreal injection of NN2101 substantially inhibited the SCF/cKIT-mediated choroidal neovascularization (CNV), with efficacy comparable to aflibercept, a vascular endothelial growth factor inhibitor. A combined intravitreal injection of NN2101 and aflibercept resulted in an additive therapeutic effect on CNV. NN2101 neither caused ocular toxicity nor interfered with the early retinal vascular development in mice. Ocular pharmacokinetic analysis in rabbits indicated that NN2101 demonstrated a pharmacokinetic profile suitable for intravitreal injection. These findings provide the first evidence of the potential use of the anti-cKIT blocking antibody, NN2101, as an alternative or additive therapeutic for the treatment of neovascular AMD.