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Background: Antibody drug conjugates (ADCs) and immunotoxins (ITs) are promising anticancer immunotherapeutics. Despite their encouraging performance in clinical trials, both ADCs and ITs often suffer from disadvantages such as stoichiometrically undefined chemical linkage of the cytotoxic payload (ADCs) and the potential immunogenicity of toxins derived from bacteria and plants (ITs). Methods: Human microtubule-associated protein tau (MAP) was cloned in-frame with human EGF, expressed in E. coli and purified by standard chromatographic methods. The in vitro activity was confirmed by flow cytometry, cell viability assays and tubulin polymerisation assay. The in vivo efficacy was demonstrated using noninvasive far-red in vivo imaging. Results: The EGF-MAP selectively induced apoptosis in EGFR-overexpressing proliferating cancer cells through stabilisation of microtubules. Nonproliferating cells were not affected, demonstrating superior selectivity of EGF-MAP for cancer cells. The EGF-MAP was well tolerated at high doses in mice compared with the ETA'-based control. The in vivo efficacy of EGF-MAP was demonstrated in a tumour xenograft mouse model. Conclusion: Our data indicate the general mechanism of action for a new class of human immunotherapeutic reagents suitable for the treatment of cancer. This approach combines the binding specificity of targeting ligands with the selective cytotoxicity of MAP towards proliferating cells.

Over the past few years, the SNAP-tag technology has become a methodology with great potential in a variety of applications, e.g. the (specific) visualization of individual proteins and studies of protein interaction in living cells. Furthermore, the tag can be used for immunopurification and detection of recombinant proteins or site-specific coupling of recombinant proteins to surfaces. Next to the in vitro applications, it also enables detection of tagged proteins in vivo. This review gives an overview of the SNAP-tag technology in different fields of research and its potential for future developments.

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.

Preclinical in vivo analyses of treatment responses are an important prerequisite to evaluate new therapeutics. Molecular in vivo imaging in the far red (FR)/near infra red (NIR) is a promising method, as it enables measurements at different time points in individual animals, thereby reducing the number of animals required, while increasing statistical significance. Here, we show the establishment of a method to monitor response to treatment using fluorescent cells, expressing the epidermal growth factor receptor (EGFR), a target already used in therapy.We transfected A-431 tumour cells with the far red-emitting protein Katushka (Kat2), resulting in strong fluorescence allowing for the monitoring of tumour growth when implanted in BALB/c nu/nu mice with a CRi Maestro in vivo imager. We targeted A-431 cells with a previously reported immunotoxin (IT), consisting of the anti-EGFR antibody single-chain variable fragment (scFv) 425, fused to Pseudomonas aeruginosa Exotoxin A' (ETA'). In addition, EGFR expression was verified using the 425(scFv) conjugated to a NIR dye BG-747 through a SNAP-tag linker.The results show the feasibility to evaluate response to treatment in vivo by FR imaging, while at the same location detecting EGFR expression. Treatment with 425(scFv)-ETA' resulted in decelerated tumour growth, while not affecting the overall health of the animals. This is in contrast to treatment with Doxorubicin, which, although decreasing the tumour size, resulted in poor health.We developed a novel method to non-invasively determine treatment responses by in vivo imaging of multiple parameters which showed the efficacy of 425(scFv)-ETA'.

Chondroitin sulfate proteoglycan 4 (CSPG4) has been identified as a highly promising target antigen for immunotherapy of triple-negative breast cancer (TNBC). TNBC represents a highly aggressive heterogeneous group of tumors lacking expression of estrogen, progesterone and human epidermal growth factor receptor 2. TNBC is particularly prevalent among young premenopausal women. No suitable targeted therapies are currently available and therefore, novel agents for the targeted elimination of TNBC are urgently needed. Here, we present a novel cytolytic fusion protein (CFP), designated αCSPG4(scFv)-MAP, that consists of a high affinity CSPG4-specific single-chain antibody fragment (scFv) genetically fused to a functionally enhanced form of the human microtubule-associated protein (MAP) tau. Our data indicate that αCSPG4(scFv)-MAP efficiently targets CSPG4(+) TNBC-derived cell lines MDA-MB-231 and Hs 578T and potently inhibits their growth with IC50 values of ∼200 nM. Treatment with αCSPG(scFv)-MAP resulted in induction of the mitochondrial stress pathway by activation of caspase-9 as well as endonuclease G translocation to the nucleus, while induction of the caspase-3 apoptosis pathway was not detectable. Importantly, in vivo studies in mice bearing human breast cancer xenografts revealed efficient targeting to and accumulation of αCSPG4(scFv)-MAP at tumor sites resulting in prominent tumor regression. Taken together, this preclinical proof of concept study confirms the potential clinical value of αCSPG4(scFv)-MAP as a novel targeted approach for the elimination of CSPG4-positive TNBC.

Theranostics 4(10), 960-971 (2014). doi:10.7150/thno.9293, Published by Ivyspring, Wyoming, NSW

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 intraand extracellular compartments of living cells. The fusion proteins were coupled to SNAP-surface(®) Alexa Fluor(®) 488 or SNAP-surface(®) Alexa Fluor(®) 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.