Abstract 5176: A novel, completely human fusion construct containing active Granzyme B efficiently kills Folate Receptor alpha positive tumor cells

Abstract

Abstract Folate receptor alpha (FRα) is an attractive therapeutic target because it is highly overexpressed on several tumors such as ovarian, breast and lung cancers and demonstrates low or restricted distribution on normal tissues. In addition, FRα has been shown to play a tumor promoting role with its expression associated with poor prognosis of cancer patients. In our laboratory, we have generated two new molecules targeting FRα and containing active granzyme B (GrB) as the cytotoxic payload. We used as binding domains two different scFvs targeting the FRα from antibodies used in clinical trials. These human scFvs (designated mov003 and mov018) were fused to human GrB through an engineered IgG heavy-chain fragment (designated as Fc) which contains a dimerization domain. The constructs were cloned into the pSECTag vector containing an upstream 5 kDa purification tag with an enterokinase (EK) cleavage site for facile removal. The molecules were expressed in transiently-transfected HEK-293E suspension cells over 3 days at 37°C in 5% CO2. The molecules were isolated from culture media and purified by ion-exchange chromatography followed by EK cleavage to remove the free tag. The yields were 12-18 mg/L for both molecules. The assessment of the specific binding of each molecule to the antigen target is ongoing. The cytotoxicity of the constructs against a panel of cell lines expressing various levels of FRα showed a specific cytotoxic effect with an IC50 in the low nanomolar range (5-100 nM), particularly exhibiting very low IC50 values against breast (MDA-MB-231) and ovarian cell lines (IGROV-1). Receptor negative cells demonstrated IC50 levels in the micromolar range. Based on IC50 values, these data demonstrate suggest the fusion constructs targeting FRα are efficiently internalized and effectively deliver the active serine protease to the cytosol, activating the cytotoxic cascade. In vitro stability assays over 96 hours showed that both constructs are highly stable in mouse serum. Immunofluorescence and confocal studies to examine the time course of internalization and the pro-apoptotic events triggered by these agents are under examination and will be presented. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Ana Alvarez de Cienfuegos, Laurence H. Cheung, Khalid A. Mohamedali, Walter N. Hittelman, Michael G. Rosenblum. A novel, completely human fusion construct containing active Granzyme B efficiently kills Folate Receptor alpha positive tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5176.