CUSP06, a Novel CDH6-Targeted Antibody-Drug Conjugate, Demonstrates Antitumor Efficacy in Multiple CDH6-Expressing Human Cancer Models

Abstract

Background/Objectives: Cadherin-6 (CDH6), also known as K-cadherin, is a type II classic cadherin molecule that plays an important role in the embryonic development of the kidney but has very limited expression in adult tissues. It is overexpressed in several human malignancies, primarily in ovarian cancer, renal cell carcinoma, as well as, less frequently, cholangiocarcinoma, uterine serous carcinoma, glioma, lung, pancreatic and thyroid cancers. The characteristic of limited expression in normal tissues, high expression in tumor tissues, and rapid internalization upon antibody binding makes CDH6 a well-suited antibody-drug conjugate (ADC) target. Methods: We developed a novel CDH6-targeting ADC, CUSP06, consisting of a proprietary humanized antibody selective for CDH6, a protease cleavable linker, and an exatecan payload, with a drug-to-antibody ratio (DAR) of 8. We further characterized the pharmacological activities of CUSP06 in multiple in vitro and in vivo models. Results: CUSP06 was selectively bound to cell surface CDH6 and was efficiently internalized into CDH6-positive ovarian cancer cells, and led to the induction of DNA damage and apoptosis of CDH6-positive cancer cells. CUSP06 exhibited strong antiproliferative activity against several CDH6-positive cancer cell lines and demonstrated strong bystander cell killing effect in the cell mixing experiments in vitro. CUSP06 exhibits excellent in vivo antitumor efficacy in CDH6-high or -low cell line-derived xenograft (CDX) or patient-derived xenograft (PDX) models from human ovarian, renal and uterine cancers, as well as cholangiocarcinoma. CUSP06 demonstrated a favorable safety profile in GLP-compliant toxicology studies in Sprague Dawley rats and cynomolgus monkeys. Conclusions: The preclinical data highlighted the therapeutic potential of CUSP06 in multiple CDH6-positive human cancers.

Keywords: ADC (antibody-drug conjugate); CDH6; K-cadherin; cadherin-6; cholangiocarcinoma; exatecan; ovarian cancer; uterine cancer.

Figure 1

Structure, property and selectivity of CUSP06. (A) Schematic structure of CUSP06. (B) Size exclusion chromatogram of CUSP06 indicates CUSP06 is homogenous in solution. (C) In vitro plasma stability of CUSP06 in buffer (PBST + 1% BSA), human, rat and monkey plasma. (D) Binding affinity of CUSP06 to human, monkey, rat and mouse CDH6. (E) Binding selectivity of CUSP06 for human CDH6, CDH9 and CDH10 by ELISA.

Figure 1

Structure, property and selectivity of CUSP06. (A) Schematic structure of CUSP06. (B) Size exclusion chromatogram of CUSP06 indicates CUSP06 is homogenous in solution. (C) In vitro plasma stability of CUSP06 in buffer (PBST + 1% BSA), human, rat and monkey plasma. (D) Binding affinity of CUSP06 to human, monkey, rat and mouse CDH6. (E) Binding selectivity of CUSP06 for human CDH6, CDH9 and CDH10 by ELISA.

Figure 2

In vitro Characterization of CUSP06. (A) Internalization of CUSP06 in two ovarian cancer cell lines, OVCAR3 and PA-1. (B) Determination of DNA damage and apoptosis caused by CUSP06 in OVCAR3 cells. After OVCAR-3 cells were treated with CUSP06, CUSP06 mAb, IgG-ADC control or exatecan for 72 h, pChk1, total Chk1, p-H2AX, cleaved PARP, and β-actin level were detected by Western Blot. (C) Characterization of the antiproliferative activities of CUSP06 in CDH6-positive (OVCAR3 and PA-1) and CDH6-null ES2 cells by CCK8 assay. (D) Characterization of in vitro bystander effect of CUSP06. A mixture of OVCAR3 cells and ES-2-GFP cells were treated with 1.25 nM IgG-ADC, CUSP06, or R-DXd for 5 days. The viable cell numbers were determined by FACS analysis.

Figure 2

In vitro Characterization of CUSP06. (A) Internalization of CUSP06 in two ovarian cancer cell lines, OVCAR3 and PA-1. (B) Determination of DNA damage and apoptosis caused by CUSP06 in OVCAR3 cells. After OVCAR-3 cells were treated with CUSP06, CUSP06 mAb, IgG-ADC control or exatecan for 72 h, pChk1, total Chk1, p-H2AX, cleaved PARP, and β-actin level were detected by Western Blot. (C) Characterization of the antiproliferative activities of CUSP06 in CDH6-positive (OVCAR3 and PA-1) and CDH6-null ES2 cells by CCK8 assay. (D) Characterization of in vitro bystander effect of CUSP06. A mixture of OVCAR3 cells and ES-2-GFP cells were treated with 1.25 nM IgG-ADC, CUSP06, or R-DXd for 5 days. The viable cell numbers were determined by FACS analysis.

Figure 3

In vivo antitumor activity of CUSP06 in CDX models. (A) Antitumor activity of CUSP06 in OVCAR3 xenograft model. A single dose of 10 mg/kg of CUSP06 was administered intravenously to the tumor-bearing mice at Day 0. Each data represents mean and SEM of tumor volume or relative body weight changes. Each group contained six mice. (B) Antitumor activity of CUSP06 in PA-1 xenograft model. A single dose of 2.5 or 5 mg/kg CUSP06 was administered intravenously to the tumor-bearing mice at Day 0. Each data represents mean and SEM of tumor volume or relative body weight changes. Each group contained five mice. (C) Antitumor activity of CUSP06 in 786-O xenograft model. Two doses of 10 mg/kg of CUSP06 or IgG-T1000-e were administered intravenously to the tumor-bearing mice at Day 0 and D21 of the study. Each data represents mean and SEM of tumor volume or relative body weight changes. Each group contained five mice.

Figure 4

In vivo antitumor activity of CUSP06 in PDX models. (A) Antitumor activity of CUSP06 in a CDH6-high Ovarian PDX model (LD-1588). A single dose of 10 mg/mg CUSP06 or IgG-ADC control was administered intravenously to the tumor-bearing mice at Day 0. Each data represents mean and SEM of tumor volume or relative body weight changes. Each group contained five mice. (B) Antitumor activity of CUSP06 in a CDH6-low Ovarian PDX model (LD-2851). A single dose of 3 mg/mg CUSP06 or IgG-ADC control was administered intravenously to the tumor-bearing mice at Day 0. Each data represents mean and SEM of tumor volume or relative body weight changes. Each group contained five mice. (C) Antitumor activity of CUSP06 in a CDH6-low kidney nephroblastoma PDX model (LD-2511). A single dose of 10 mg/mg CUSP06, R-DXd, R-T1000-e, or IgG-ADC control was administered intravenously to the tumor-bearing mice at Day 0. Each data represents mean and SEM of tumor volume or relative body weight changes. Each group contained five mice. (D) Antitumor activity of CUSP06 in a CDH6-low cholangiocarcinoma PDX model (LD-2214). A single dose of 10 mg/mg CUSP06, or IgG-ADC control was administered intravenously to the tumor-bearing mice at Day 0. Each data represents mean and SEM of tumor volume or relative body weight changes. Each group contained five mice. (E) Antitumor activity of CUSP06 in a CDH6-medium uterine cancer PDX model (UT3705). A single dose of 3 or 10 mg/mg CUSP06, or IgG-ADC control was administered intravenously to the tumor-bearing mice on Day 0. Each data represents mean and SEM of tumor volume or relative body weight changes. Each group contained five mice.

Figure 4

In vivo antitumor activity of CUSP06 in PDX models. (A) Antitumor activity of CUSP06 in a CDH6-high Ovarian PDX model (LD-1588). A single dose of 10 mg/mg CUSP06 or IgG-ADC control was administered intravenously to the tumor-bearing mice at Day 0. Each data represents mean and SEM of tumor volume or relative body weight changes. Each group contained five mice. (B) Antitumor activity of CUSP06 in a CDH6-low Ovarian PDX model (LD-2851). A single dose of 3 mg/mg CUSP06 or IgG-ADC control was administered intravenously to the tumor-bearing mice at Day 0. Each data represents mean and SEM of tumor volume or relative body weight changes. Each group contained five mice. (C) Antitumor activity of CUSP06 in a CDH6-low kidney nephroblastoma PDX model (LD-2511). A single dose of 10 mg/mg CUSP06, R-DXd, R-T1000-e, or IgG-ADC control was administered intravenously to the tumor-bearing mice at Day 0. Each data represents mean and SEM of tumor volume or relative body weight changes. Each group contained five mice. (D) Antitumor activity of CUSP06 in a CDH6-low cholangiocarcinoma PDX model (LD-2214). A single dose of 10 mg/mg CUSP06, or IgG-ADC control was administered intravenously to the tumor-bearing mice at Day 0. Each data represents mean and SEM of tumor volume or relative body weight changes. Each group contained five mice. (E) Antitumor activity of CUSP06 in a CDH6-medium uterine cancer PDX model (UT3705). A single dose of 3 or 10 mg/mg CUSP06, or IgG-ADC control was administered intravenously to the tumor-bearing mice on Day 0. Each data represents mean and SEM of tumor volume or relative body weight changes. Each group contained five mice.

Figure 5

Concentration of CUSP06 (A), total antibody (B) and free exatecan (C) in monkey plasma after the first intravenous administration of CUSP06 at 10, 20, and 30 mg/kg from monkey toxicology study. Plasma concentrations of CUSP06 and total antibody were determined by ligand-binding assay. Plasma concentration of free exatecan was determined by LC-MS. Each value represents the mean and SD (N = 10). The concentration of free exatecan at 504 h after administration of 20 mg/kg CUSP06 is below LLOQ and is recorded as 0 ng/mL.