Bystander killing effect of DS-8201a, a novel anti-human epidermal growth factor receptor 2 antibody-drug conjugate, in tumors with human epidermal growth factor receptor 2 heterogeneity

Abstract

Antibody-drug conjugates deliver anticancer agents selectively and efficiently to tumor tissue and have significant antitumor efficacy with a wide therapeutic window. DS-8201a is a human epidermal growth factor receptor 2 (HER2)-targeting antibody-drug conjugate prepared using a novel linker-payload system with a potent topoisomerase I inhibitor, exatecan derivative (DX-8951 derivative, DXd). It was effective against trastuzumab emtansine (T-DM1)-insensitive patient-derived xenograft models with both high and low HER2 expression. In this study, the bystander killing effect of DS-8201a was evaluated and compared with that of T-DM1. We confirmed that the payload of DS-8201a, DXd (1), was highly membrane-permeable whereas that of T-DM1, Lys-SMCC-DM1, had a low level of permeability. Under a coculture condition of HER2-positive KPL-4 cells and negative MDA-MB-468 cells in vitro, DS-8201a killed both cells, whereas T-DM1 and an antibody-drug conjugate with a low permeable payload, anti-HER2-DXd (2), did not. In vivo evaluation was carried out using mice inoculated with a mixture of HER2-positive NCI-N87 cells and HER2-negative MDA-MB-468-Luc cells by using an in vivo imaging system. In vivo, DS-8201a reduced the luciferase signal of the mice, indicating suppression of the MDA-MB-468-Luc population; however, T-DM1 and anti-HER2-DXd (2) did not. Furthermore, it was confirmed that DS-8201a was not effective against MDA-MB-468-Luc tumors inoculated at the opposite side of the NCI-N87 tumor, suggesting that the bystander killing effect of DS-8201a is observed only in cells neighboring HER2-positive cells, indicating low concern in terms of systemic toxicity. These results indicated that DS-8201a has a potent bystander effect due to a highly membrane-permeable payload and is beneficial in treating tumors with HER2 heterogeneity that are unresponsive to T-DM1.

Keywords: Antibody-drug conjugate; HER2; T-DM; bystander killing; topoisomerase I inhibitor.

Figure 1

Structures of antibody–drug conjugates (ADCs) and released payloads. DXd, DX‐8951 derivative; HER2, human epidermal growth factor receptor 2; T‐DM1, trastuzumab emtansine.

Figure 2

Bystander killing effect of antibody–drug conjugates (ADCs) in coculture conditions in vitro. (a) Growth inhibitory activity of ADCs against KPL‐4 and MDA‐MB‐468 cells. Tumor cells were treated with ADCs for 5 days and cell viability (%) was calculated. Each point represents the mean and SD (n = 3). KPL‐4 and MDA‐MB‐468 cells were cocultured and treated with 10 nM ADCs for 5 days. After collecting adherent cells, cell number and ratio of human epidermal growth factor receptor 2 (HER2)‐positive and HER2‐negative cells were determined by a cell counter and a flow cytometer, respectively. (b) Data of flow cytometric analysis. (c) Numbers of KPL‐4 and MDA‐MB‐468 viable cells. Each bar represents the mean and SD (n = 3). DXd, DX‐8951 derivative; T‐DM1, trastuzumab emtansine.

Figure 3

Antitumor activity of antibody–drug conjugates (ADCs) and model establishment for bystander evaluation. (a) Antitumor activity of ADCs in NCI‐N87 and MDA‐MB‐468‐Luc xenograft models. The mean estimated tumor volume and SE (n = 6) are represented on the graph. (b) Human epidermal growth factor receptor 2 (HER2) expression in co‐inoculated tumor. Eight days after s.c. inoculation, each tumor was collected and fixed with 10% neutral‐buffered formalin. Immunohistochemistry of HER2 protein was carried out using the HercepTest II kit according to manufacturer's instructions. DXd, DX‐8951 derivative; T‐DM1, trastuzumab emtansine.

Figure 4

Bystander killing in co‐inoculated conditions in vivo. Seven days after inoculation of a mixture of both NCI‐N87 and MDA‐MB‐468‐Luc cells, mice were i.v. given antibody–drug conjugates (day 0). Luciferase activity was detected by in vivo imager after i.v. administration of substrate. (a) Imaging data of luciferase activity. (b) Luciferase activity. (c) Estimated tumor volume. SE (n = 5) represented on the graph. DXd, DX‐8951 derivative; HER2, human epidermal growth factor receptor 2; T‐DM1, trastuzumab emtansine.

Figure 5

Bystander killing effect on distant tumor. Nude mice were s.c. inoculated with a mixture of both NCI‐N87 and MDA‐MB‐468‐Luc cells into the right flank, and only MDA‐MB‐468‐Luc into the left flank. Seven days after inoculation, mice were treated with i.v. DS‐8201a at 3 mg/kg. Luciferase activity was detected by an in vivo imager after i.v. administration of substrate. (a) Imaging data of luciferase activity. (b) Mean luciferase activity and SE (n = 5) are represented on the graph.