A novel anti‐c‐Kit antibody–drug conjugate to treat wild‐type and activating‐mutant c‐Kit‐positive tumors

Abstract

c-Kit overexpression and activating mutations, which are reported in various cancers, including gastrointestinal stromal tumor (GIST), small-cell lung cancer (SCLC), acute myeloid leukemia, acral melanoma, and systemic mastocytosis (SM), confer resistance to tyrosine kinase inhibitors (TKIs). To overcome TKI resistance, an anti-c-Kit antibody-drug conjugate was developed in this study to treat wild-type and mutant c-Kitpositive cancers. NN2101, a fully human IgG1, was conjugated to DM1, a microtubule inhibitor, through N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) (to give NN2101-DM1). The antitumor activity of NN2101-DM1 was evaluated in vitro and in vivo using various cancer cell lines. NN2101-DM1 exhibited potent growth-inhibitory activities against c-Kit-positive cancer cell lines. In a mouse xenograft model, NN2101-DM1 exhibited potent growth-inhibitory activities against imatinib-resistant GIST and SM cells. In addition, NN2101-DM1 exhibited a significantly higher anti-cancer effect than carboplatin/etoposide against SCLC cells where c-Kit does not mediate cancer pathogenesis. Furthermore, the combination of NN2101-DM1 with imatinib in imatinib-sensitive GIST cells induced complete remission compared with treatment with NN2101-DM1 or imatinib alone in mouse xenograft models. These results suggest that NN2101-DM1 is a potential therapeutic agent for wild-type and mutant c-Kit-positive cancers.

Antibody-Drug Conjugate Targeting c-Kit for the Treatment of Small Cell Lung Cancer

“…Interestingly, 4C9 antibody binding was saturated at 50 ng/mL in c-Kit-positive SCLC cell lines. In addition, the expression of c-Kit was higher in NCI-H889 cells compared with that in NCI-H526 and NCI-H1048 cells, which is consistent with a previous report [34]. However, 4C9 did not show cross-reactivity with NCI-H446 and NCI-H2170 cells, which are c-Kit-negative SCLC cell lines.…”

“…4C9 antibody (2 ng/mL) was then added to each well at RT for 1 h. The plate was washed with 1 × PBS containing 0.1% Tween-20 four times and incubated with HRP-conjugated anti-human IgG secondary antibody (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. Then, 3, 3 , 5, 5 -tetramethylbenzidine solution (Thermo Fisher Scientific, Waltham, MA, USA) was added for 5 min. The signal was measured at 450 nm using a SPECTROstar Nano Microplate Reader (BMG LABTECH, Ortenberg, GermanyELISA was also used to compare the binding affinities of 4C9 and 4C9-DM1 as previously described [34].…”

“…The internalization assay was carried out as previously described [34]. Briefly, the cells were incubated with cycloheximide (75 µg/mL) to prevent the transport of de novo synthesized c-Kit to the cytoplasmic membrane.…”

A fully human anti-c-Kit monoclonal antibody 2G4 inhibits proliferation and degranulation of human mast cells

“…In previous studies, we demonstrated that 2G4 and 4C9 antibodies bind to c-Kit with high binding a nity (K D of 2G4 = 2.83 × 10 − 12 M and K D of 4C9 = 5.58 × 10 − 9 M). Moreover, 2G4 antibody, as an antagonist of c-Kit, inhibited SCF/c-Kit signaling in various cancer cell lines [34,35]. In this study, the human mast cell line LAD2, which highly expresses FcεRI and c-Kit (Supplementary Fig.…”

“…To overcome the limitations of anti-IgE antibodies, we aimed to develop therapeutic antibody that can inhibit both the proliferation and degranulation of mast cells. Previous studies have demonstrated that 2G4 antibody, a fully human antibody, could inhibit the activation of SCF/c-Kit signaling in various cancer cell lines [34,35]. In addition, another anti-c-Kit antibody 4C9 signi cantly reduced the expression of c-Kit [36].…”