Preclinical Efficacy of the Antibody-Drug Conjugate CLDN6-23-ADC for the Treatment of CLDN6-Positive Solid Tumors

Abstract

Purpose: Claudin-6 (CLDN6) is expressed at elevated levels in multiple human cancers including ovarian and endometrial malignancies, with little or no detectable expression in normal adult tissue. This expression profile makes CLDN6 an ideal target for development of a potential therapeutic antibody-drug conjugate (ADC). This study describes the generation and preclinical characterization of CLDN6-23-ADC, an ADC consisting of a humanized anti-CLDN6 monoclonal antibody coupled to monomethyl auristatin E (MMAE) via a cleavable linker.

Experimental design: A fully humanized anti-CLDN6 antibody was conjugated to MMAE resulting in the potential therapeutic ADC, CLDN6-23-ADC. The antitumor efficacy of CLDN6-23-ADC was assessed for antitumor efficacy in CLDN6-positive (CLDN6+) and -negative (CLDN6-) xenografts and patient-derived xenograft (PDX) models of human cancers.

Results: CLDN6-23-ADC selectively binds to CLDN6, versus other CLDN family members, inhibits the proliferation of CLDN6+ cancer cells in vitro, and is rapidly internalized in CLDN6+ cells. Robust tumor regressions were observed in multiple CLDN6+ xenograft models and tumor inhibition led to markedly enhanced survival of CLDN6+ PDX tumors following treatment with CLDN6-23-ADC. IHC assessment of cancer tissue microarrays demonstrate elevated levels of CLDN6 in 29% of ovarian epithelial carcinomas. Approximately 45% of high-grade serous ovarian carcinomas and 11% of endometrial carcinomas are positive for the target.

Conclusions: We report the development of a novel ADC, CLDN6-23-ADC, that selectively targets CLDN6, a potential onco-fetal-antigen which is highly expressed in ovarian and endometrial cancers. CLDN6-23-ADC exhibits robust tumor regressions in mouse models of human ovarian and endometrial cancers and is currently undergoing phase I study.

Figure 1.

CLDN6 expression in human cancer tissues and cell lines. A, Expression of CLDN6 by RNA transcript in the TCGA dataset of cancerous tissue samples. Individual patient samples are depicted with box and whisker plots showing the median, 25th and 75th percentiles of expression. B, Expression of CLDN6 by RNA transcript in a panel of 500+ human cancer cell lines. Individual cell lines are depicted with box and whisker plots showing the median, 25th and 75th percentiles of expression. C, Immunoblot of CLDN6 protein expression in cancer cell lines, with beta actin as a loading control.

Figure 2.

Binding of a humanized CLDN6 antibody to CLDN6+ cell line models. A, Binding of CLDN6–23-mAb to CLDN6+ cell line models with CLDN6–23-mAb primary labelled with AF647 (red) and Hoechst 33342 nuclear stain (blue). Flow binding of CLDN6–23-mAb compared with human IgG is also depicted. All cells were treated with 20 μg/mL of CLDN6–23-mAb or human IgG. B, Binding of CLDN6–23-mAb (10 μg/mL) in artificial cell lines overexpressing CLDN3, CLDN4, CLDN6, or CLDN9 by flow cytometry.

Figure 3.

In vivo efficacy, ADCC activity, and internalization of humanized CLDN6 antibody. Efficacy of CLDN6–23-mAb compared with human IgG control in CLDN6+ xenografts; (A) UMUC4 (10 mg/kg every 4 days), (B) H841 (10 mg/kg once weekly), and (C) OV90 (10 mg/kg once weekly). D, Efficacy of CLDN6–23-mAb in CLDN6− M202 xenograft (10 mg/kg once weekly) compared with human IgG control. In all xenografts, lines represent mean tumor volume ± SEM and P values from repeated measures ANOVA are depicted as: *, P < 0.05; **, P < 0.001; ***, P < 0.0001, or n.s., not significant. E,In vitro ADCC response assay with CLDN6–23-mAb in ARK2 and M202 cells compared with human IgG control. F, UMUC4 xenografts established in NSG mice treated with CLDN6–23-mAb (10 mg/kg once weekly), where lines represent mean tumor volume ± SEM and P values from repeated measures ANOVA are depicted as: *, P < 0.05; **, P < 0.001; ***, P < 0.0001, or n.s., not significant. G, ARK2 cells transfected with LAMP1 (green) were stained with Hoechst 33342 (blue) and treated with CLDN6–23-mAb [labelled with AF647 (red)]. Yellow regions indicate colocalization of CLDN6 and LAMP1.

Figure 4.

Internalization and efficacy of anti-CLDN6 ADC. A, CLDN6–23-ADC and CLDN6–23-mAb binding by flow cytometry in CLDN6+ and CLDN6− cell lines. B, ARK2 and OVCA429 cells transfected with LAMP1 (green) were stained with Hoechst 33342 (blue) and treated with CLDN6–23-ADC [labelled with AF647 (red)]. Yellow regions indicate colocalization of CLDN6 and LAMP1. C, 2D proliferation assays with OVCA429 (left) and M202 (right) cells following treatment with a range of concentrations of CLDN6–23-ADC (0–333.33 nmol/L). D,In vivo efficacy of CLDN6–23-ADC (5 mg/kg, intravenously once weekly) compared with CLDN6–23-mAb (10 mg/kg intravenously once weekly) in UMUC4 xenografts where lines represent mean tumor volume ± SEM and P values from pairwise t tests are depicted as: *, P < 0.05; **, P < 0.001; ***, P < 0.0001, or n.s., not significant. E, H&E stains of tumors excised from UMUC4 xenografts on day 3 and day 11 following treatment with either CLDN6–23-mAb (10 mg/kg) or CLDN6–23-ADC (5 mg/kg).

Figure 5.

Sustained responses to anti-CLDN6 ADC treatment. Efficacy of CLDN6–23-ADC in CLDN6+ UMUC4 xenografts (A), ARK2 xenografts (B), and OV90 (C): UMUC4 2.5 mg/kg intravenously once weekly, ARK2 5.0 mg/kg intravenously once weekly, OV90 2.5 mg/kg intravenously once weekly; all for three treatments only, where lines represent mean tumor volume ± SEM and P values from repeated measures ANOVA are depicted as: *, P < 0.05; **, P < 0.001; ***, P < 0.0001, or n.s., not significant. D, CLDN6 expression in panel of ovarian PDXs. Two CLDN6+ models (DF149 and DF181) and one CLDN6− model (DF20) were selected for in vivo assessment based on the immunoblot shown. E, Tumor burden, as measured by bioluminescence imaging. F, Survival curves for NSG mice bearing ovarian PDXs following treatment with either CLDN6–23-mAb (10 mg/kg) or CLDN6–23-ADC (5 mg/kg) intravenously once weekly.