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Aachen, Germany

Pardo A.,RWTH Aachen | Stocker M.,RWTH Aachen | Kampmeier F.,RWTH Aachen | Melmer G.,Pharmedartis GmbH | And 4 more authors.
Cancer Immunology, Immunotherapy | Year: 2012

Purpose 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. Methods 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. Results 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. Conclusions 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'. © Springer-Verlag 2012.

Bialon M.,RWTH Aachen | Schellenberg L.,RWTH Aachen | Herzog N.,RWTH Aachen | Kraus S.,RWTH Aachen | And 13 more authors.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy | Year: 2014

Monoclonal antibodies are produced in cultured hybridoma cell lines, but these cells tend to be unstable; it is therefore necessary to rescue the corresponding genetic information. Here we describe an improved method for the amplification of antibody variable gene (V-gene) information from murine hybridoma cells using a panel of specific, non-degenerate primers. This primer set allows sequences to be rescued from all murine V-genes, except the lambda light chain genes, which rarely contribute to murine immune diversity. We tested the primers against a range of antibodies and recovered specific amplification products in all cases. The heavy and light chain variable regions were subsequently joined by a two-step cloning strategy or by splice overlap extension PCR. © Copyright 2014, Mary Ann Liebert, Inc.

Pharmedartis Gmbh | Date: 2012-09-21

The present disclosure provides novel variants of enzymes exhibiting serine protease activity; nucleic acid molecules encoding said proteases, vectors, host cells containing the nucleic acids and methods for preparation and producing such enzymes; compositions and complexes comprising at least one of the proteases; and methods for using such enzymes as a part of an immunoprotease, in particular for the treatment of cancer.

Schiffer S.,RWTH Aachen | Schiffer S.,Fraunhofer Institute for Molecular Biology and Applied Ecology | Hansen H.P.,University of Cologne | Hehmann-Titt G.,Pharmedartis GmbH | And 5 more authors.
Blood Cancer Journal | Year: 2013

Tumors develop when infiltrating immune cells contribute growth stimuli, and cancer cells are selected to survive within such a cytotoxic microenvironment. One possible immune-escape mechanism is the upregulation of PI-9 (Serpin B9) within cancer cells. This serine proteinase inhibitor selectively inactivates apoptosis-inducing granzyme B (GrB) from cytotoxic granules of innate immune cells. We demonstrate that most classical Hodgkin lymphoma (cHL)-derived cell lines express PI-9, which protects them against the GrB attack and thereby renders them resistant against GrB-based immunotherapeutics. To circumvent this disadvantage, we developed PI-9-insensitive human GrB mutants as fusion proteins to target the Hodgkin-selective receptor CD30. In contrast to the wild-type GrB, a R201K point-mutated GrB construct most efficiently killed PI-9-positive and -negative cHL cells. This was tested in vitro and also in vivo whereby a novel optical imaging-based tumor model with HL cell line L428 was applied. Therefore, this variant, as part of the next generation immunotherapeutics, also named cytolytic fusion proteins showing reduced immunogenicity, is a promising molecule for (targeted) therapy of patients with relapsing malignancies, such as cHL, and possibly other PI-9-positive malignancies, such as breast or lung carcinoma.© 2013 Macmillan Publishers Limited All rights reserved.

Hristodorov D.,RWTH Aachen | Amoury M.,RWTH Aachen | Mladenov R.,RWTH Aachen | Niesen J.,Pharmedartis GmbH | And 12 more authors.
Molecular Cancer Therapeutics | Year: 2014

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, wefurther 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. © 2014 AACR.

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