Visualisierung und Analysen von Therapieansätzen durch fluoreszenzbasierte molekulare Bildgebungsverfahren

Preclinical in vivo analyses of therapeutic efficacy are an important prerequisite for the evaluation of new therapeutics. Optical molecular imaging in the far red or NIR range is a favourable method, since it allows measurements at different time points in one single animal. This not only reduces the amount of animals required per experiment, but in addition increases statistical significance. A431 cells have a very high expression of the epidermal growth factor receptor (EGFR). This receptor is also strongly over-expressed by a variety of tumors and thus qualifies as a target for therapeutic intervention. However, a well-known problem in EGFR-specific therapy is the down-regulation of the receptor. The aim of this work was the visualisation of therapeutic effects by the use of optical molecular imaging. For that, mainly 3 different aspects were tested. On one hand, tumor cells should be used, that allow the monitoring of cell growth during therapy in vivo on the basis of their inherent fluorescence. Furthermore, the induced apoptosis should be detectable using fluorescently labeled Annexin A5. Finally, after therapy receptor expression should be verified using a receptor-specific construct to exclude the possibility of development of therapy resistance due to down-regulation of the receptor. For the in vivo experiments, A431 tumor cells, that have been passaged in mice before, were transfected with the far red fluorescent protein Katushka (Kat 2). One in-house developed immunotoxin, consisting of a scFv targeted against the EGFR, fused to a deletion mutant of Pseudomonas aeruginosa Exotoxin A, showed specific binding and high toxicity to A431 cells. Fluorescent A431scM3-Kat2 cells were injected subcutaneously in vivo. After tumor development, treatment was executed with the immunotoxin or with the chemotherapeutic Doxorubicin. Tumor sizes were determined on basis of the cell-inherent fluorescence at different time points. Furthermore, tumor-specific EGFR-expression was assessed through binding of the same 425(scFv) coupled to a NIR dye. Treatment with the immunotoxin 425(scFv)-ETA’ led to a decelerated tumor growth without having an impact on the overall health of the mice, this unlike the chemotherapeutic Doxorubicin. The here described method allows the simultaneous assessment of several parameters in one single animal, like tumor growth and receptor expression. This is particularly important for the in vivo evaluation of new therapeutics and can be employed to monitor treatment of different types of tumors

Visualisierung und Analysen von Therapieansätzen durch fluoreszenzbasierte molekulare Bildgebungsverfahren

Preclinical in vivo analyses of therapeutic efficacy are an important prerequisite for the evaluation of new therapeutics. Optical molecular imaging in the far red or NIR range is a favourable method, since it allows measurements at different time points in one single animal. This not only reduces the amount of animals required per experiment, but in addition increases statistical significance. A431 cells have a very high expression of the epidermal growth factor receptor (EGFR). This receptor is also strongly over-expressed by a variety of tumors and thus qualifies as a target for therapeutic intervention. However, a well-known problem in EGFR-specific therapy is the down-regulation of the receptor. The aim of this work was the visualisation of therapeutic effects by the use of optical molecular imaging. For that, mainly 3 different aspects were tested. On one hand, tumor cells should be used, that allow the monitoring of cell growth during therapy in vivo on the basis of their inherent fluorescence. Furthermore, the induced apoptosis should be detectable using fluorescently labeled Annexin A5. Finally, after therapy receptor expression should be verified using a receptor-specific construct to exclude the possibility of development of therapy resistance due to down-regulation of the receptor. For the in vivo experiments, A431 tumor cells, that have been passaged in mice before, were transfected with the far red fluorescent protein Katushka (Kat 2). One in-house developed immunotoxin, consisting of a scFv targeted against the EGFR, fused to a deletion mutant of Pseudomonas aeruginosa Exotoxin A, showed specific binding and high toxicity to A431 cells. Fluorescent A431scM3-Kat2 cells were injected subcutaneously in vivo. After tumor development, treatment was executed with the immunotoxin or with the chemotherapeutic Doxorubicin. Tumor sizes were determined on basis of the cell-inherent fluorescence at different time points. Furthermore, tumor-specific EGFR-expression was assessed through binding of the same 425(scFv) coupled to a NIR dye. Treatment with the immunotoxin 425(scFv)-ETA’ led to a decelerated tumor growth without having an impact on the overall health of the mice, this unlike the chemotherapeutic Doxorubicin. The here described method allows the simultaneous assessment of several parameters in one single animal, like tumor growth and receptor expression. This is particularly important for the in vivo evaluation of new therapeutics and can be employed to monitor treatment of different types of tumors

Novel PSCA targeting scFv-fusion proteins for diagnosis and immunotherapy of prostate cancer

Purpose Despite great progress in the diagnosis and treatment of localized prostate cancer (PCa), there remains a need for new diagnostic markers that can accurately distinguish indolent and aggressive variants. One promising approach is the antibody-based targeting of prostate stem cell antigen (PSCA), which is frequently overexpressed in PCa. Here, we show the construction of a molecular imaging probe comprising a humanized scFv fragment recognizing PSCA genetically fused to an engineered version of the human DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT), the SNAP-tag, enabling specific covalent coupling to various fluorophores for diagnosis of PCa. Furthermore, the recombinant immunotoxin (IT) PSCA(scFv)-ETA' comprising the PSCA(scFv) and atruncated version of Pseudomonas exotoxin A (PE, ETA') was generated. Methods We analyzed the specific binding and internalization behavior of the molecular imaging probe PSCA(scFv)SNAP in vitro by flow cytometry and live cell imaging, compared to the corresponding IT PSCA(scFv)-ETA'. The cytotoxic activity of PSCA(scFv)-ETA' was tested using cell viability assays. Specific binding was confirmed on formalin-fixed paraffin-embedded tissue specimen of early and advanced PCa. Results Alexa -Fluor (R) 647 labeling of PSCA(scFv)-SNAP confirmed selective binding to PSCA, leading to rapid internalization into the target cells. The recombinant IT PSCA(scFv)-ETA' showed selective binding leading to internalization and efficient elimination of target cells. Conclusions Our data demonstrate, for the first time, the specific binding, internalization, and cytotoxicity of a scFv-based fusion protein targeting PSCA. Immunohistochemical staining confirmed the specific ex vivo binding to primary PCa material

Microtubule-associated protein tau facilitates the targeted killing of proliferating cancer cells in vitro and in a xenograft mouse tumour model in vivo

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

In vitro effects and ex vivo binding of an EGFR-specific immunotoxin on rhabdomyosarcoma cells

PURPOSE: Rhabdomyosarcoma (RMS) is a rare and aggressive soft tissue sarcoma with limited treatment options and a high failure rate during standard therapy. New therapeutic strategies based on targeted immunotherapy are therefore much in demand. The epidermal growth factor receptor (EGFR) has all the characteristics of an ideal target. It is overexpressed in up to 80 % of embryonal RMS and up to 50 % of alveolar RMS tumors. We therefore tested the activity of the EGFR-specific recombinant immunotoxin (IT) 425(scFv)-ETA' against EGFR(+) RMS cells in vitro and ex vivo. METHODS: We tested the specific binding and internalization behavior of 425(scFv)-ETA' in RMS cell lines in vitro by flow cytometry, compared to the corresponding imaging probe 425(scFv)-SNAP monitored by live cell imaging. The cytotoxic activity of 425(scFv)-ETA' was tested using cell viability and apoptosis assays. Specific binding of the IT was confirmed on formalin-fixed paraffin-embedded tissue samples from two RMS patients. RESULTS: We confirmed the specific binding of 425(scFv)-ETA' to RMS cells in vitro and ex vivo. Both the IT and the corresponding imaging probe were rapidly internalized. The IT killed EGFR(+) RMS cells in a dose-dependent manner, while showing no effect against control cells. It showed specific apoptotic activity against one selected RMS cell line. CONCLUSIONS: This is the first study showing the promising therapeutic potential of a recombinant, EGFR-targeting, ETA'-based IT on RMS cells. We confirmed the selective killing with IC50 values of up to 50 pM, and immunohistochemical staining confirmed the specific ex vivo binding to primary RMS material

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

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