EpCAM and CSPG4 scFv based fusion proteins for the treatment of triple negative breast cancer

Triple-negative breast cancer (TNBC) defines a heterogeneous group of breast tumors lacking the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). It has the poorest prognosis of all breast cancer subtypes and high prevalence among young premenopausal women. Unlike other breast cancer subtypes, no effective targeted immunotherapy is currently available. Due to the heterogeneity of TNBC, no single receptor can be expected to be effective in targeting all TNBCs. Therefore, in this study, we set out to develop a panel of receptors for targeted therapies which can, together, include the majority of TNBC tumors. Through a literature search two markers stood out as suitable targets, EpCAM- overexpressed in 60% of TNBC and CSPG4- overexpressed in 20-40% of TNBC. EpCAM and CSPG4 are differentially expressed and their expression on TNBC tumor does not overlap. Therefore, novel single-chain variable fragment (scFv)-based human cytolytic fusion proteins (hCFPs) were constructed to target these receptors. Their activity and mechanism of action were compared to corresponding recombinant immunotoxins (ITs) utilizing the Pseudomonas Exotoxin A- deletion mutant (ETA’) as the toxic moiety. To target EpCAM expressing TNBC, a hCFP designated Gb-R201K-αEpCAM(scFv) was generated, in which an EpCAM-selective scFv was genetically fused to a human Granzyme B (Gb) mutant with reduced sensitivity to its natural inhibitor serpin B9. To target CSPG4 a CFP designated αCSPG4(scFv)-MAP was used, consisting of a CSPG4-selective scFv genetically fused to a functionally enhanced form of the human microtubule-associated protein (MAP) tau. In vitro studies confirmed specific binding, internalization and subsequent potent cytotoxic activity of both hCFPs, with IC50 values of 200-300 nM for Gb-R201K-αEpCAM(scFv) towards TNBC cell lines MDA-MB-453 and MDA-MB-468 and 200-400nM for αCSPG4(scFv)-MAP toward MDA-MB-231 and Hs 578T. In vivo studies in subcutaneous tumor models revealed that treatment of mice bearing MDA-MB-468 tumors with Gb-R201K-αEpCAM(scFv) inhibited tumor growth of up to 50 %. Treatment of mice bearing MDA-MB-231 tumors with αCSPG4(scFv)-MAP resulted in prominent inhibition of tumor growth and to tumor regression of more than 50%.Taken together, αCSPG4(scFv)-MAP and Gb-R201K-αEpCAM(scFv) appear to be clinically relevant and promising novel targeted agent for the treatment of TNBC

SNAP-tag based Agents for Preclinical In Vitro Imaging in Malignant Diseases

Although current cancer treatment strategies are highly aggressive, they are often not effective enough to destroy the collectivity of malignant cells. The residual tumor cells that survived the first-line treatment may continue to proliferate or even metastasize. Therefore, the development of novel more effective strategies to specifically eliminate also single cancer cells is urgently needed. In this respect, the development of antibody-based therapeutics, in particular example immunotoxins, has attracted broad interest. Since the internalization of immunotoxins is essential for their cytotoxic effectivity, it is of crucial importance to study their internalization behavior to assess the potential for their therapeutic use. In this study, we determined the internalization behavior of four different single-chain fragments variable (scFv) when binding to the corresponding target antigen as expressed on solid or non-solid tumor cell lines. The scFvs were recombinantly fused to the SNAP-tag, an engineered variant of the human repair enzyme O-6-alkylguanine-DNA alkyltransferase that covalently reacts with benzylguanine derivatives. Since a large number of highly sensitive organic fluorescent dyes are already available or can easily be derivatized to react with the self-labeling SNAP-tag, this system provides versatile applications for imaging of intra- and extracellular compartments of living cells. The fusion proteins were coupled to SNAP-surface (R) Alexa Fluor (R) 488 or SNAP-surface (R) Alexa Fluor (R) 647 and binding as well as internalization was monitored by flow cytometry and confocal microscopy, respectively. Depending on the respective target antigen, we could distinguish between slow and rapid internalization behavior. Moreover, we detected increased internalization rate for bivalent scFv constructs. Our approach allows for rapid and early stage evaluation of the internalization characteristics of new antibodies designated for further therapeutic development

EpCAM-Selective Elimination of Carcinoma Cells by a Novel MAP-Based Cytolytic Fusion Protein

In normal epithelia, the epithelial cell adhesion molecule (EpCAM) expression is relatively low and only present at the basolateral cell surface. In contrast, EpCAM is aberrantly overexpressed in various human carcinomas. Therefore, EpCAM is considered to be a highly promising target for antibody-based cancer immunotherapy. Here, we present a new and fully human cytolytic fusion protein (CFP), designated “anti–EpCAM(scFv)-MAP,” that is comprised of an EpCAM-specific antibody fragment (scFv) genetically fused to the microtubule-associated protein tau (MAP). Anti–EpCAM(scFv)-MAP shows potent EpCAM-restricted proapoptotic activity toward rapidly proliferating carcinoma cells. In vitro assays confirmed that treatment with anti–EpCAM(scFv)-MAP resulted in the colocalization and stabilization of microtubules, suggesting that this could be the potential mode of action. Dose-finding experiments indicated that anti–EpCAM(scFv)-MAP is well tolerated in mice. Using noninvasive far-red in vivo imaging in a tumor xenograft mouse model, we further demonstrated that anti–EpCAM(scFv)-MAP inhibited tumor growth in vivo. In conclusion, our data suggest that anti–EpCAM(scFv)-MAP may be of therapeutic value for the targeted elimination of EpCAM+ carcinomas

Granzyme B-based cytolytic fusion protein targeting EpCAM specifically kills triple negative breast cancer cells in vitro and inhibits tumor growth in a subcutaneous mouse tumor model

Triple-negative breast cancer (TNBC) is associated with poor prognosis and high prevalence among young premenopausal women. Unlike in other breast cancer subtypes, no targeted therapy is currently available. Overexpression of epithelial cell adhesion molecule (EpCAM) in 60% of TNBC tumors correlates with poorer prognosis and is associated with cancer stem cell phenotype. Thus, selective elimination of EpCAM(+) TNBC tumor cells is of clinical importance. Therefore, we constructed a fully human targeted cytolytic fusion protein, designated GbR201K-alpha EpCAM(scFv), in which an EpCAM-selective single-chain antibody fragment (scFv) is genetically fused to a granzyme B (Gb) mutant with reduced sensitivity to its natural inhibitor serpin B9. In vitro studies confirmed its specific binding, internalization and cytotoxicity toward a panel of EpCAM-expressing TNBC cells. Biodistribution kinetics and tumor-targeting efficacy using MDA-MB-468 cells in a human TNBC xenograft model in mice revealed selective accumulation of GbR201K-aEpCAM(scFv) in the tumors after i.v. injection. Moreover, treatment of tumor-bearing mice demonstrated a prominent inhibition of tumor growth of up to 50 % in this proof-of-concept study. Taken together, our results indicate that GbR201K-alpha EpCAM(scFv) is a promising novel targeted therapeutic for the treatment of TNBC. (C) 2016 Elsevier Ireland Ltd. All rights reserved