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

Abstract

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 "antiEpCAM(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 þ carcinomas. Mol Cancer Ther; 13(9); 2194-202. Ó2014 AACR.

Human MAP tau based targeted cytolytic fusion proteins

“…The anti-CD30 single-chain antibody fragment Ki4(scFv) fused in frame to MAP tau for treatment of CD30 + malignancies such as Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (sALCL) was shown to induce apoptosis in rapidly proliferating L540cy, L428, and Karpas 299 cells in a dose-dependent manner with IC50 values of ~53 nmol/l, which is comparabllike to that obtained (IC50 36 nmol/L) for the Gb_R201K-Ki4(scFv), a granzyme B-derived mutant. Also, an antiEpCAM(scFv)-MAP fusion protein showed promising results with IC50 values of 43 nmol/L and 67 nmol/L against L3.6pl and A431 EpCAM + cancer cell lines, which is also comparably better when compared with that obtained for anti-EpCAM(scFv)-ETA' (409 nmol/L on L3.6pl and 91 nmol/L on A431 [53]. As earlier described, it is important to note that differences in IC50 values are attributed to different target antigens and as well as their differences in expression on different cells and cell lines.…”

“…Hetzel et al, in 2008 reported the significant contribution of small cleavable adapter sequences to enhancing the cytotoxicity of human cytolytic fusion proteins. Indeed the use of these adapter sequence is a major aim in our future MAP-tau based preclinical studies [53].…”

Generation of an artificial human B cell line test system using Transpo-mAbTM technology to evaluate the therapeutic efficacy of novel antigen-specific fusion proteins

“…To overcome this challenge, a new generation of immunotoxins has been developed containing human pro-apoptotic proteins such as granzyme B, angiogenin or MAPTau [21, 27–30, 54, 60, 61]. We therefore used the established pMS vector system [53] to prepare fusion constructs combining two human effector proteins with TTC as a model antigen, resulting in two novel TTC-based fusion proteins: EGrB(R201K)-TTC and TTC-MAPTau.…”

“…The yield of TTC-MAPTau was 1.6 mg/l, which is comparable to other MAPTau fusion proteins such as Ki4scFv-MAP and anti-EpCAMscFv-MAP produced in E . coli with yields of up to 1 mg/l [60, 63, 64]. Eukaryotic expression systems offer the advantage of endotoxin-free fusion protein production, so that additional purification steps are unnecessary.…”

“…Cells with high proliferation rates, such as cancer cells, are more sensitive to MAPTau, as demonstrated in earlier studies testing MAPTau fusion proteins as candidates for cancer therapy. For example, EGF-MAP achieved a half-maximal inhibitory concentration (IC 50 ) of 1 μM against pancreatic L3.6pI cells, and Ki(scFv)-MAP achieved an IC 50 value of 53 nM against the Hodgkin’s lymphoma cell line L540cy [60, 63, 64]. Autoreactive memory B cells are also characterized by slow proliferation, so MAPTau fusion proteins may be less useful than granzyme B fusion proteins in this context.…”