A Novel Antibody-Drug Conjugate Targeting Nectin-2 Suppresses Ovarian Cancer Progression in Mouse Xenograft Models

Abstract

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 (K_D = 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.

Keywords: antibody-drug conjugate; chimeric antibody; nectin-2; ovarian cancer.

Figure 1

Characterization of the chimeric 12G1 (c12G1) antibody. (A) The binding of c12G1 antibody was determined using FACS analysis, at the indicated concentrations. Daudi cells were used as the nectin-2-negative cell line. (B) HEK293 cells were transfected with 40 nM of control or nectin-2 si-RNA, for 72 h. The FACS analysis was then conducted as described in the Methods section (*, vs. control si-RNA). Knock-down of nectin-2 expression was confirmed using western blot. Tubulin was used as the loading control. Anti-nectin-2 antibody from Abcam was used for western blot. The red arrowhead indicates a non-specific band. This experiment was independently repeated at least three times. (C) The binding affinity of c12G1 antibody to human nectin-2 was examined using SPR analysis. * p < 0.05.

Figure 2

Identification of the nectin-2-binding domain of c12G1. (A) PVRIG (100 ng/well) was coated to 96-well plates and the binding of nectin-2 (1 µg/mL) to PVRIG was investigated in the presence of c12G1 antibody, at the indicated concentrations. The results represent mean ± SD of three independent experiments. (B) Schematic diagram for the construction of nectin-2 deletion mutants. The extracellular domain of the indicated nectin-2 deletion constructs was cloned into the pCMV6 FLAG vector. (C) The wild-type and deletion nectin-2 mutants were transfected into HEK293 cells. The whole cell lysates obtained from these cells were then subjected to immunoprecipitation assay using c12G1 antibody, followed by western blot with an anti-FLAG antibody. (D) To determine whether c12G1 antibody can bind to endogenous nectin-2 protein, the whole cell lysates of non-transfected HEK293 cells were subjected to immunoprecipitation assay using the c12G1 antibody or normal human IgG, followed by western blot with an anti-nectin-2 antibody. Ten percent (10 µg) of the cell lysates were loaded as the input control. Tubulin was used as the loading control. All experiments were independently repeated at least three times. HC indicates heavy chain of antibody.

Figure 3

The c12G1 antibody is internalized into ovarian cancer cells. (A) Ovarian cancer cells were incubated in the presence or absence of c12G1 (1 µg/mL) at 4 °C or 37 °C, for 1–3 h, and then subjected to flow cytometer analysis. The fluorescence signal of the c12G1-nectin-2 complex on the cell surface decreased after incubation at 37 °C. (B) OV-90, SK-OV-3, or Caov-3 cells were treated with the c12G1/Zenon™-conjugated anti-Fc FAB complex for 15 h, following which the Zenon™-positive cells were analyzed using FACS (* and ***, vs. control). All experiments were independently repeated at least three times. * p < 0.05, *** p < 0.0001.

Figure 4

Characterization of c12G1-DM1. (A) The naked c12G1 antibody and c12G1-DM1 were compared using non-reducing and reducing SDS-PAGE. (B) The optical absorbance of c12G1-DM1 at the wavelength of 252 nm was compared to that of the naked c12G1 antibody. The calculated DAR was determined as 5.07. The binding affinity of naked c12G1 antibody and c12G1-DM1 to human nectin-2 protein was compared using ELISA (C) and flow cytometry (D). The nectin-2-binding affinities of naked c12G1 antibody and c12G1-DM1 were similar. Daudi cells were used as the nectin-2-negative cell line. All experiments were independently repeated at least three times.

Figure 5

c12G1-DM1 exhibited anti-tumor activity, both in vitro and in vivo. (A) Ovarian cancer cells were treated with vehicle, c12G1 antibody (1 µg/mL), IgG-DM1 (1 µg/mL), or c12G1-DM1 (1 µg/mL), for 24 and 48 h. The cells were then fixed and stained with propidium iodide, followed by cell cycle analysis using a Celigo Imaging Cytometer (*, **, and ***, vs. their respective corresponding vehicle, c12G1, and IgG-DM1). c12G1-DM1 increased the cell population in the G2/M phase at 24 h, followed by an increase in the G1 population at 48 h. Daudi cells were used as the nectin-2-negative cell line. The results have been represented as mean ± SD from at least three independent experiments. (B) Cells were treated with serially diluted concentrations of the c12G1 antibody or c12G1-DM1, for 3–4 d. The cells were stained with 10 µM Hoechst 33342, at 37 °C for 30 min, and quantified using a Celigo Imaging Cytometer. The results represent mean ± SD of at least three independent experiments. The dashed line indicates the number of seeded cells. (C) Ovarian cancer cell lines were implanted into immunodeficient mice. The mice were randomized into different treatment groups when the tumor volume reached ~200 mm³ (n = 6), and then intravenously administered vehicle, c12G1, IgG-DM1, or c12G1-DM1, as indicated. Green arrows indicate the administration of vehicle, c12G1, IgG-DM1, or c12G1-DM1 (# and ##, vs. their respective vehicle, c12G1, and IgG-DM1). * p < 0.01, ** p < 0.001, *** p < 0.0001, ^# p < 0.01, and ^## p < 0.001.