Abstract 4937: Targeting epithelial membrane protein 2 on breast tumor cells with a fusion construct containing the serine protease granzyme B
Authors: Mohamedali Khalid , Mohandessi Shabnam , Cheung Lawrence , Rosenblum Michael G. , Wadehra Madhuri 
Published: August 2018

Despite significant advances in biology and medicine, the incidence and mortality due to breast cancer world-wide is still unacceptably high. Conventional anticancer therapeutics often suffer from lack of specificity, resulting in poor therapeutic indexes and substantial toxicities to normal healthy tissues. Immunoconjugates represent an innovative therapeutic approach to deliver a deadly payload to a tumor cell with more specificity and potentially less avenues for resistance than either can exert individually. However, it has been a challenge to find candidates as well as implement conjugation strategies to achieve these goals for a number of cancers. Epithelial membrane protein-2 (EMP2) is over-expressed in a number of gynecological cancers, including in 63% of invasive breast cancers and in 100% of ALDH+ breast cancer stem cells. Clinically, EMP2 is a biomarker for disease severity and progression in these cancers as its expression has been linked to characteristics of advanced disease including increased tissue invasion and accelerated growth. We have recently developed an IgG1 targeting the extracellular domain of EMP2 with single digit nM affinity. Administration of the anti-EMP2 IgG1 was successful at reducing breast cancer tumor load. As the antibody-antigen complex rapidly internalizes, we sought to improve on the efficacy of the naked antibody. Granzyme B (GrB) is a serine protease that plays a critical role in the body's defense against viral infection and tumor development by initiating the apoptotic cascade via both caspase-dependent and -independent mechanisms. Granzyme B-containing fusion proteins can circumvent MDR-1 mediated multi-drug resistance when delivered to targeted cancer cells. Accordingly, we developed novel fusion proteins composed of the anti-EMP2 backbone as the targeting moiety and granzyme B as the cytotoxic payload. The designs comprised (a) the anti-EMP2 IgG1 with GrB fused to the Fc domain through a cleavable linker, and (b) active GrB fused to the IgG heavy chain (Fc) domain containing 2 single-chain antibodies against EMP2, a format that results in a longer plasma retention time. The constructs were expressed by transient transfection in HEK293E cells and purified to homogeneity. Flow cytometry and ELISA demonstrated binding properties of the fusion protein identical to the naked antibody. The enzymatic activity of the fusion construct was similar to commercially-available GrB. The constructs were highly cytotoxic to EMP2-positive cells with superior in vitro efficacy over the naked antibody (10 nM vs 50nM, respectively), compared to EMP2-negative cells. Moreover, these agents show minimum toxicity and high anti-tumor potency. We predict that these designs may improve therapeutic outcomes for EMP2+ tumors. Research conducted, in part, by the Clayton Foundation for Research (K.M.) and by R01 CA163971 (M. W.).

Citation Format: Khalid Mohamedali, Shabnam Mohandessi, Lawrence Cheung, Michael G. Rosenblum, Madhuri Wadehra. Targeting epithelial membrane protein 2 on breast tumor cells with a fusion construct containing the serine protease granzyme B [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4937.