Abstract 6314: Novel granzyme B-based fusion constructs targeting FRα on liquid and solid tumors
Authors: Alvarez-Cienfuegos Ana , Cheung Lawrence H. , Mohamedali Khalid A. , Baran Natalia , Konopleva Marina , Rosenblum Michael G. 
Published: June 2023

Abstract
Folate receptor α (FRα) has been an attractive therapeutic target for many decades because it is highly overexpressed on several solid tumors and demonstrates low or restricted distribution on healthy tissues. Additionally, high expression of FRα has been associated with tumor progression and thus poor prognosis of cancer patients. Therefore, utilizing FRα to effectively locate tumor cells for targeted therapy using antibody drug conjugates, small molecule drug conjugates, radioimmunoconjugates or chimeric antigen receptor T cells, has been extensively investigated. Although some of the mentioned targeted therapies have shown promising preclinical results, their clinical utility has been limited due to associated toxicities in healthy tissues. We generated two new fusion constructs targeting FRα and containing active human granzyme B (GrB) as the cytotoxic payload. We used as binding domains two different scFvs targeting the FRα from antibodies previously used in clinical trials. These human scFvs (mov003 and mov018) were fused to the human serine protease GrB through an engineered human IgG heavy-chain fragment (designated as Fc) which contains a dimerization domain for longer half-life. 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 and ovarian cell lines. FRα negative cells demonstrated IC50 levels in the micromolar range. Immunofluorescence studies demonstrated that the fusion constructs targeting FRα are efficiently internalized and effectively delivered the active serine protease to the cytosol. In vitro stability assays over 96 hours showed that both constructs are highly stable in mouse serum. Additionally, recent publications suggested that FRα is significantly associated with acute myeloid leukemia (AML) and might serve as a therapeutic target. We assessed the cytotoxic efficacy of GrB-based constructs on a panel of FRα+ AML cell lines and have found that these cell lines were very sensitive (IC50 in the low nanomolar range). Furthermore, evaluation of GrB fusion efficacy in two AML cell lines resistant to Bcl-2 inhibitor Venetoclax showed IC50s to be similar or lower than their parental cell lines. These results, therefore, demonstrate no cross-resistance to GrB. The safe and effective profile of these drugs together with their unique mechanism of action makes them ideal candidates for advancing pre-clinical development and may offer a potential new strategy for treatment of solid tumors and particularly Venetoclax relapsed AML patients, ineligible for intensive chemotherapy. In vivo efficacy studies to compare the two constructs are warranted and will help to identify the optimal therapeutic for further clinical consideration. Research conducted, in part, by the Clayton Foundation for Research.
Citation Format: Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Natalia Baran, Marina Konopleva, Michael G. Rosenblum. Novel granzyme B-based fusion constructs targeting FRα on liquid and solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6314.