Ovarian cancer is the fifth leading cause of cancer, followed by front line is mostly platinum agents and PARP inhibitors, and very limited option in later lines. Therefore, there is a need for alternative therapeutic options. Nectin-2, which is overexpressed in ovarian cancer, is a known immune checkpoint that deregulates immune cell function. In this study, we generated a novel anti-nectin-2 antibody (chimeric 12G1, c12G1), and further characterized it using epitope mapping, enzyme-linked immunosorbent assay, surface plasmon resonance, fluorescence-activated cell sorting, and internalization assays. The c12G1 antibody specifically bound to the C2 domain of human nectin-2 with high affinity (KD = 2.90 × 10-10 M), but not to mouse nectin-2. We then generated an antibody-drug conjugate comprising the c12G1 antibody conjugated to DM1 and investigated its cytotoxic effects against cancer cells in vitro and in vivo. c12G1-DM1 induced cell cycle arrest at the mitotic phase in nectin-2-positive ovarian cancer cells, but not in nectin-2-negative cancer cells. c12G1-DM1 induced ~100-fold cytotoxicity in ovarian cancer cells, with an IC50 in the range of 0.1 nM~7.4 nM, compared to normal IgG-DM1. In addition, c12G1-DM1 showed ~91% tumor growth inhibition in mouse xenograft models transplanted with OV-90 cells. These results suggest that c12G1-DM1 could be used as a potential therapeutic agent against nectin-2-positive ovarian cancers.
Immunoconjugates , Maytansine , Ovarian Neoplasms , Humans , Mice , Animals , Female , Immunoconjugates/pharmacology , Immunoconjugates/therapeutic use , Heterografts , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Platinum/pharmacology , Xenograft Model Antitumor Assays , Cell Line, Tumor , Cell Proliferation , Carcinoma, Ovarian Epithelial/drug therapy , Ovarian Neoplasms/pathology , Immunoglobulin G/pharmacology , Immunoglobulin G/therapeutic use , Maytansine/therapeutic use
Lung cancer is the leading cause of cancer-related deaths. Small cell lung cancer (SCLC) accounts for 15-25% of all lung cancers. It exhibits a rapid doubling time and a high degree of invasiveness. Additionally, overexpression of c-Kit occurs in 70% of SCLC patients. In this study, we evaluated an antibody-drug conjugate (ADC) that targets c-Kit, which is a potential therapeutic agent for SCLC. First, we generated and characterized 4C9, a fully human antibody that targets c-Kit and specifically binds to SCLC cells expressing c-Kit with a binding affinity of KD = 5.5 × 10-9 M. Then, we developed an ADC using DM1, a microtubule inhibitor, as a payload. 4C9-DM1 efficiently induced apoptosis in SCLC with an IC50 ranging from 158 pM to 4 nM. An in vivo assay using a xenograft mouse model revealed a tumor growth inhibition (TGI) rate of 45% (3 mg/kg) and 59% (5 mg/kg) for 4C9-DM1 alone. Combination treatment with 4C9-DM1 plus carboplatin/etoposide or lurbinectedin resulted in a TGI rate greater than 90% compared with the vehicle control. Taken together, these results indicate that 4C9-DM1 is a potential therapeutic agent for SCLC treatment.
Antibodies, Monoclonal, Humanized/pharmacology , Immunoconjugates/pharmacology , Lung Neoplasms/drug therapy , Small Cell Lung Carcinoma/drug therapy , Animals , Carboplatin/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Etoposide/pharmacology , Female , Humans , Lung Neoplasms/metabolism , Maytansine/pharmacology , Mice , Proto-Oncogene Proteins c-kit/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, ErbB-2/metabolism , Small Cell Lung Carcinoma/metabolism , Trastuzumab/pharmacology , Tubulin Modulators/metabolism , Xenograft Model Antitumor Assays/methods
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 (KD = 2.92 × 10-10 M), rats (KD = 1.68 × 10-6 M), mice (KD = 11.5 × 10-9 M), and humans (KD = 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.
Antibodies, Neutralizing/immunology , Antineoplastic Agents, Immunological/immunology , Proto-Oncogene Proteins c-kit/immunology , Animals , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/pharmacology , Antineoplastic Agents, Immunological/chemistry , Antineoplastic Agents, Immunological/pharmacology , Binding Sites, Antibody , Cell Proliferation/drug effects , Cells, Cultured , Epitopes/chemistry , Epitopes/immunology , HEK293 Cells , Haplorhini , Humans , Mice , Proto-Oncogene Proteins c-kit/antagonists & inhibitors , Rats