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PHARMEDARTIS GmbH | Date: 2016-10-11

A polypeptide having a serine protease variant of the human granzyme B set forth by SEQ ID NO: 1, wherein the serine protease variant has at least 95% identity to SEQ ID NO: 1 and has a substitution at the position that corresponds structurally or by amino acid sequence homology to position Arg201 of SEQ ID NO: 1, and wherein the serine protease variant has activity to cleave the motif Ile-Glu-Thr-Asp.


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.


PubMed | University of Groningen, Fraunhofer Institute for Molecular Biology and Applied Ecology, RWTH Aachen and Pharmedartis GmbH
Type: Journal Article | Journal: 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, 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.


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.


Schiffer S.,RWTH Aachen | Schiffer S.,Fraunhofer Institute for Molecular Biology and Applied Ecology | Rosinke R.,Fraunhofer Institute for Molecular Biology and Applied Ecology | Jost E.,RWTH Aachen | And 6 more authors.
International Journal of Cancer | Year: 2014

CMML (chronic myelomonocytic leukemia) belongs to the group of myeloid neoplasms known as myelodysplastic and myeloproliferative diseases. In some patients with a history of CMML, the disease transforms to acute myelomonocytic leukemia (AMML). There are no specific treatment options for patients suffering from CMML except for supportive care and DNA methyltransferase inhibitors in patients with advanced disease. New treatment strategies are urgently required, so we have investigated the use of immunotherapeutic directed cytolytic fusion proteins (CFPs), which are chimeric proteins comprising a selective domain and a toxic component (preferably of human origin to avoid immunogenicity). The human serine protease granzyme B is a prominent candidate for tumor immunotherapy because it is expressed in cytotoxic T lymphocytes and natural killer cells. Here, we report the use of CD64 as a novel target for specific CMML and AMML therapy, and correlate CD64 expression with typical surface markers representing these diseases. We demonstrate that CD64-specific human CFPs kill CMML and AMML cells ex vivo, and that the mutant granzyme B protein R201K is more cytotoxic than the wild-type enzyme in the presence of the granzyme B inhibitor PI9. Besides, the human CFP based on the granzyme B mutant was also able to kill AMML or CMML probes resistant to Pseudomonas exotoxin A. What's new? New treatment strategies for chronic (CMML) and acute myelomonocytic leukemia (AMML) are urgently needed. This study suggests that CD64 is not only a promising diagnostic marker but also a novel target for specific CMML and AMML therapy. The data show that CD64-specific human cytolytic fusion proteins kill CMML and AMML cells ex vivo, with the mutant granzyme B protein R201K being more cytotoxic than its wild type in the presence of the granzyme B inhibitor PI-9. GbR201K-H22(scFv) also shows superior activity to a state-of-the-art anti-CD64 immunotoxin based on Pseudomonas exotoxin A on PI-9-positive and negative primary CMML and AMML cells. Copyright © 2014 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.


Niesen J.,Fraunhofer Institute for Molecular Biology and Applied Ecology | Stein C.,Fraunhofer Institute for Molecular Biology and Applied Ecology | Stein C.,RWTH Aachen | Brehm H.,RWTH Aachen | And 8 more authors.
Journal of Cancer Research and Clinical Oncology | Year: 2015

Purpose: The epidermal growth factor receptor (EGFR) is overexpressed in many solid tumors. EGFR-specific monoclonal antibodies (mAbs), such as cetuximab and panitumumab, have been approved for the treatment of colorectal and head and neck cancer. To increase tissue penetration, we constructed single-chain fragment variable (scFv) antibodies derived from these mAbs and evaluated their potential for targeted cancer therapy. The resulting scFv-based EGFR-specific immunotoxins (ITs) combine target specificity of the full-size mAb with the cell-killing activity of a toxic effector domain, a truncated version of Pseudomonas exotoxin A (ETA′). Methods: The ITs and corresponding imaging probes were tested in vitro against four solid tumor entities (rhabdomyosarcoma, breast, prostate and pancreatic cancer). Specific binding and internalization of the ITs scFv2112-ETA′ (from cetuximab) and scFv1711-ETA′ (from panitumumab) were demonstrated by flow cytometry and for the scFv-SNAP-tag imaging probes by live cell imaging. Cytotoxic potential of the ITs was analyzed in cell viability and apoptosis assays. Binding of the ITs was proofed ex vivo on rhabdomyosarcoma, prostate and breast cancer formalin-fixed paraffin-embedded biopsies. Results: Both novel ITs showed significant pro-apoptotic and anti-proliferative effects toward the target cells, achieving IC50 values of 4 pM (high EGFR expression) to 460 pM (moderate EGFR expression). Additionally, rapid internalization and specific in vitro and ex vivo binding on patient tissue were confirmed. Conclusions: These data demonstrate the potent therapeutic activity of two novel EGFR-specific ETA′-based ITs. Both molecules are promising candidates for further development toward clinical use in the treatment of various solid tumors to supplement the existing therapeutic regimes. © 2015, Springer-Verlag Berlin Heidelberg.


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.


PubMed | Fraunhofer Institute for Molecular Biology and Applied Ecology and Pharmedartis GmbH
Type: Journal Article | Journal: Cancer letters | Year: 2016

Human cytolytic fusion proteins (hCFPs) offer a promising immunotherapeutic approach for the treatment of solid tumors, avoiding the immunogenicity and undesirable side-effects caused by immunotoxins derived from plants or bacteria. The well-characterized human serine protease granzyme B has already been used as a therapeutic pro-apoptotic effector domain. We therefore developed a novel recombinant hCFP (GbR201K-scFv1711) consisting of an epidermal growth factor receptor-specific human antibody fragment and a granzyme B point mutant (R201K) that is insensitive to serpin B9 (PI9), a natural inhibitor of wild-type granzyme B that is often expressed in solid tumors. We found that GbR201K-scFv1711 selectively bound to epidermoid cancer and rhabdomyosarcoma cells and was rapidly internalized by them. Nanomolar concentrations of GbR201K-scFv1711 achieved the specific killing of epidermoid cancer cells by inducing apoptosis, and similar effects were observed in rhabdomyosarcoma cells when GbR201K-scFv1711 was combined with the endosomolytic substance chloroquine. The novel hCFP was stable in serum and bound to human rhabdomyosarcoma tissue ex vivo. These data confirm that GbR201K-scFv1711 is a promising therapeutic candidate suitable for further clinical investigation.


PubMed | Fraunhofer Institute for Molecular Biology and Applied Ecology, RWTH Aachen and Pharmedartis GmbH
Type: Journal Article | Journal: Journal of cancer research and clinical oncology | Year: 2015

The epidermal growth factor receptor (EGFR) is overexpressed in many solid tumors. EGFR-specific monoclonal antibodies (mAbs), such as cetuximab and panitumumab, have been approved for the treatment of colorectal and head and neck cancer. To increase tissue penetration, we constructed single-chain fragment variable (scFv) antibodies derived from these mAbs and evaluated their potential for targeted cancer therapy. The resulting scFv-based EGFR-specific immunotoxins (ITs) combine target specificity of the full-size mAb with the cell-killing activity of a toxic effector domain, a truncated version of Pseudomonas exotoxin A (ETA).The ITs and corresponding imaging probes were tested in vitro against four solid tumor entities (rhabdomyosarcoma, breast, prostate and pancreatic cancer). Specific binding and internalization of the ITs scFv2112-ETA (from cetuximab) and scFv1711-ETA (from panitumumab) were demonstrated by flow cytometry and for the scFv-SNAP-tag imaging probes by live cell imaging. Cytotoxic potential of the ITs was analyzed in cell viability and apoptosis assays. Binding of the ITs was proofed ex vivo on rhabdomyosarcoma, prostate and breast cancer formalin-fixed paraffin-embedded biopsies.Both novel ITs showed significant pro-apoptotic and anti-proliferative effects toward the target cells, achieving IC50 values of 4 pM (high EGFR expression) to 460 pM (moderate EGFR expression). Additionally, rapid internalization and specific in vitro and ex vivo binding on patient tissue were confirmed.These data demonstrate the potent therapeutic activity of two novel EGFR-specific ETA-based ITs. Both molecules are promising candidates for further development toward clinical use in the treatment of various solid tumors to supplement the existing therapeutic regimes.

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