Andreas Lingnau

Abstract 592: Improving therapeutic activity of agonistic DR5 antibodies by inducing target binding-dependent hexamer formation

Death receptor 5 (DR5) is a highly interesting tumor target based on the enhanced sensitivity of cancer cells for DR5-dependent apoptosis. In recent years, multiple therapeutic DR5 antibodies have been evaluated in the clinic for which results however have been disappointing. IgG molecules against membrane-bound targets have shown an ability to form ordered hexameric structures upon antigen binding, a process that is dependent on Fc-Fc interactions between IgG molecules. We identified specific mutations in the human IgG1 Fc domain that enhance such antigen-dependent hexamerization while retaining solution-monomericity and developability characteristics of regular IgG1 molecules (HexaBody technology). We hypothesized that antibody-mediated hexamerization, when applied to DR5-specific antibodies, would enhance DR5 signaling and apoptosis, resulting in strongly improved therapeutic potential. The technology was applied to two non-crossblocking DR5-specific IgG1 antibodies, IgG1-DR5-01 and IgG1-DR5-05, by mutating a glutamic acid residue at position 430 in the Fc domain to glycine (the HexaBody mutants were designated Hx-DR5-01 and Hx-DR5-05). Cytotoxicity of the DR5 antibodies was explored in vitro using the CellTiter-Glo luminescent cell viability assay and the Caspase-Glo 3/7 assay in a broad panel of cancer cell lines, and in vivo in xenograft models. Both Hx-DR5-01 and Hx-DR5-05 induced increased cytotoxicity compared to their wild type (WT) IgG1 counterparts. Moreover, the combination of Hx-DR5-01 and Hx-DR5-05 (referred to as Hx-DR5-01/05) was found to be more potent than either Hx-DR5-01 or Hx-DR5-05 alone, or than the combination of the WT antibodies (IC50 in BxPC3 cells 0.5 and 1.5 μg/ml; maximal cytotoxicity 91% and 25% for Hx-DR5-01/05 and WT IgG1-DR5-01/05 respectively). In contrast to wild type agonistic DR5 antibodies, tumor cell killing by Hx-DR5-01/05 was independent of secondary crosslinking. Potent anti-tumor activity was observed in seven xenograft models for multiple indications, with Hx-DR5-01/05 consistently showing significantly better efficacy than the WT DR5 comparator antibody conatumumab. The cytotoxic activity of DR5 antibodies was significantly enhanced by the introduction of a hexamer-enhancing mutation in the IgG1 Fc domain. Maximal killing activity was obtained by combining two non-crossblocking DR5 antibodies carrying this mutation (Hx-DR5-01 and Hx-DR5-05). The strong cytotoxicity of Hx-DR5-01/05 was completely dependent on target binding but, in contrast to WT antibodies, did not require secondary crosslinking. These promising pre-clinical results support the selection of Hx-DR5-01/05 for clinical development for the treatment of cancer. Citation Format: Marije B. Overdijk, Kristin Strumane, Antonio Ortiz Buijsse, Claudine Vermot-Desroches, Andreas Lingnau, Esther C.W. Breij, Janine Schuurman, Paul W.H.I. Parren. Improving therapeutic activity of agonistic DR5 antibodies by inducing target binding-dependent hexamer formation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 592.

Abstract 1222: Establishment of a reliable metastasizing syngeneic breast cancer mouse model using orthotopically implanted 4T1 cells after several rounds of isolating and reimplanting lung metastases

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Many human breast cancer cell lines have been implanted into immunodeficient mice in order to establish in vivo xenograft models, subcutaneous or orthotopic variants, in order to mimic this type of cancer as good as possible. However, especially the metastasising capabilities of these models turned out to be highly disappointing. In order to get more reliable models, breast cancer cells have often been implanted intravenously or intracardially rather than orthotopically. These models do work fine but resemble only limited parts of the complicated metastasis process. Therefore, a metastasis event originated from an orthotopically growing breast tumor seems more appropriate and displays an expanded view on the process of metastasis. In the model presented here, the murine brast cancer cell line 4T1 was transduced with fire fly luciferase and implanted orthotopically into the fat pad of female BALB/C mice. The metastasis rate in this model is known to be better than in the common human xenografts transplanted in mice, although still limited. In order to get higher metastasis rates we tried to improve this model by resecting and recultivating the metastases of the invaded lungs after orthotopic implantation of the 4T1 cells. Cultured cells were checked for in vitro luciferase activity in parallel with ex vivo analysis of the lung tissue part which was used for the isolation of the cells. After using this approach for several rounds, what we refer to as “subpopulationing”, we checked for increased metastasis properties of this newly isolated breast cancer cells. The new 4T1-M-ortho-luc cells were implanted ortotopically in parallel to the parental 4T1-luc cell line and we analysed 9 different organs ex vivo for luciferase activity. Whereas in the parental model only in lung tissue metastasis in all implanted mice (12/12) was detectable, the metastasis rate in the new model, generated by “subpopulationing”, bounced up to 4 organs with measurable metastasis in 100 % of the mice and in general to a much higher degree of metastasis in most other organs. Only in liver tissue no metastases could be detected in both models. Treatment of the mice with Doxorubicin clearly diminished tumor growth in both breast cancer models and can therefore serve as a potent positive control here. With this new 4T1-M-ortho cell line we have available a metastasing breast cancer cell line that shows reliable metastasis observed in several organs of the mouse when implanted orthotopically. This model is suitable for testing potential anti-meatstasizing compounds aginst breast cancer. In addition, since this model is established in immunocompetent mice, and is therefore also perfectly suited for approaches involving the immune system of breast cancer patients. Citation Format: Andreas Lingnau, Steffen Hoffmann, Sandra Moor, Cynthia Schaefer-Obodozie, Christoph Schaechtele. Establishment of a reliable metastasizing syngeneic breast cancer mouse model using orthotopically implanted 4T1 cells after several rounds of isolating and reimplanting lung metastases. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1222. doi:10.1158/1538-7445.AM2014-1222

Abstract B14: Characterization of an in vivo generated subpopulation of human LNCaP prostate cancer cells as an improved testosterone-dependent subcutaneous as well as orthotopic in vivo mouse xenograft model for compound testing.

Prostate cancer is one of the most frequently diagnosed cancer entities in men. It can be estimated that in 80% of all men reaching the age of 80 years prostate tumors are present. Since a depletion of testosterone in prostate cancer therapies is often performed, androgen-dependent and -independent prostate carcinoma cell lines have been widely used to establish mouse in vivo xenograft models in order to identify novel drugs helping to eliminate prostate tumors. A commercially available LNCaP cell line was shown to be androgen-dependent since addition of synthetic androgens to androgen-free culture medium was able to restore proper cell proliferation in cell culture. However, when implanted in SCID mice in order to establish a xenograft model, the take rate turned out to be very low and tumor growth was highly heterogeneously. Therefore, we resected and recultivated one of the few well-growing tumors and reimplanted the arising subpopulation subcutaneously in mice. After performing two rounds of this “subpopulationing” procedure we could generate a new cell line, LNCaP-(Z2), which was able to form tumors in mice with a take rate of nearly 100%, but still shows heterogenous tumor growth similar to other subcutaneously implanted prostate cancer cells (e.g. PC-3). In order to demonstrate an androgen dependency of our new LNCaP subpopulation model in vivo we used castrated mice and implanted them subcutaneously in the absence or presence of also subcutaneously inoculated testosterone pellets. For comparison PC-3 prostate cancer cells were also implanted subcutaeously in the presence or absence of testosterone. Whereas no influence of testosterone was detectable on the growth behavior of subcutaneous PC-3 xenograft tumors, the development of the LNCaP-(Z2) xenografts was strictly dependent on testosterone release. We then implanted fire fly luciferase expressing LNCaP-(Z2)-luc cells orthotopically into the prostate of castrated male SCID beige mice with or without testosterone supplementation to show testosterone-dependency. Finally, treatment of Bicalutamide, a typical member of the class of anti-androgens, which is frequently used in the treatment of prostate cancer, led to significant inhibition of tumor growth using our improved subpopulation LNCaP-(Z2) in vivo model. Bicalutmid and Flutamid, another anti-androgen agent, were used for treatment in the orthopical in vivo model. Both compounds showed a noticeable anti-tumoral effect at necropsy. Thus, we could demonstrate the model9s potency as a testosterone-dependent in vivo tool suitable for screening and development of novel anti-prostate cancer drugs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B14. Citation Format: Andreas Lingnau, Sandra Moor, Steffen Hoffmann, Cynthia Schaefer-Obodozie, Ulrike Leisegang, Klotzbuecher Andrea, Schaechtele Christoph. Characterization of an in vivo generated subpopulation of human LNCaP prostate cancer cells as an improved testosterone-dependent subcutaneous as well as orthotopic in vivo mouse xenograft model for compound testing. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B14.

Abstract 2788: Characterization of an in vivo generated subpopulation of human LnCaP prostate cancer cells as an improved testosterone-dependent in vivo mouse xenograft model for compound testing.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Prostate cancer is one of the most frequently diagnosed cancer entities in men. It can be estimated that in 80% of all men reaching the age of 80 years prostate tumors are present. Since a depletion of testosterone in prostate cancer therapies is often performed, androgen-dependent and -independent prostate carcinoma cell lines have been widely used to establish mouse in vivo xenograft models in order to identify novel drugs helping to eliminate prostate tumors. A commercially available LnCaP cell line was shown to be androgen-dependent since addition of synthetic androgens to androgen-free culture medium was able to restore proper cell proliferation in cell culture. However, when implanted in SCID mice in order to establish a xenograft model, the take rate turned out to be very low and tumor growth was highly heterogeneously. Therefore, we resected and recultivated one of the few well-growing tumors and reimplanted the arising subpopulation subcutaneously in mice. After performing two rounds of this “subpopulationing” procedure we could generate a new cell line, LnCaP-Z2, which was able to form tumors in mice with a take rate of nearly 100%, but still shows heterogenous tumor growth similar to other subcutaneously implanted prostate cancer cells (e.g. PC-3). In order to demonstrate an androgen dependency of our new LnCaP subpopulation model in vivo we used castrated mice and implanted them subcutaneously with two different prostate cancer cell lines in the absence or presence of also subcutaneously inoculated testosterone pellets. Whereas no influence of testosterone was detectable on the growth behavior of subcutaneous PC-3 xenograft tumors, the development of the LnCaP-Z2 xenografts was strictly dependent on testosterone release. Finally, treatment of Bicalutamide, a typical member of the class of anti-androgens, which is frequently used in the treatment of prostate cancer, led to significant inhibition of tumor growth using our improved subpopulation LnCaP-Z2 in vivo model. Thus, we could demonstrate the models potency as a testosterone-dependent in vivo tool suitable for screening and development of novel anti-prostate cancer drugs. Citation Format: Andreas Lingnau, Steffen Hoffmann, Cynthia Schaefer-Obodozie, Ulrike Leisegang, Andreas Klotzbuecher, Christoph Schaechtele. Characterization of an in vivo generated subpopulation of human LnCaP prostate cancer cells as an improved testosterone-dependent in vivo mouse xenograft model for compound testing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2788. doi:10.1158/1538-7445.AM2013-2788

Abstract 2363: Comparison and characterization of two orthotopic glioblastoma in vivo mouse models with human U87 and LN-229 cells using in vivo bioluminescence imaging and magnetic resonance imaging (MRI)

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Glioblastoma is the most common and most aggressive type of malignant brain tumors. The prognosis is poor, with a median survival time of several months only. Despite steady progress in treatment of glioblastomas this tumor entity is still not curable. Thus, there is an urgent need for the development of novel effective therapies, and for appropriate in vivo models to characterize them. Most often novel antiglioblastoma drugs are tested in subcutaneous xenograft mouse models using various human or murine glioblastoma cell lines. To generate more relevant, orthotopic brain cancer in vivo models, we stably transduced the human glioblastoma cell lines U-87 and LN-229 with fire fly luciferase suitable for in vivo bioluminescence imaging. To initiate orthotopic growth, nude mice were anesthetized and placed in a stereotactic fixation device. Through a burr hole cells were implanted intracranially using a Hamilton syringe. Once to twice a week we used bioluminescence imaging to continually monitor tumor growth in vivo. However, as magnetic resonance imaging (MRI) is the most relevant tool to monitor oncological therapy in glioblastomas clinically, we adapted this translational aspect and analysed the mice in parallel using a dedicated animal MRI System. MRI offers detailed information about growth characteristics, size, blood supply and interaction with the surrounding tissue of the tumor. To achieve this, we performed T2-weighted imaging, and diffusion weighted imaging (DWI) weekly to visualise the tumors as well as necrosis and edemas. The use of both methods allowed independent and complementary characterization of the tumors. When exponential growth of the orthotopic tumors was observed, and the tumors reached critical sizes necropsy was done. In vivo bioluminescence imaging revealed only a very dominant signal in the region of the brain where the primary tumor was located. To address potential metastases in more detail we analysed a set of different resected organs and tissues for in vitro luciferase activity followed by extensive histological examination. Currently, we test different anti-glioblastoma agents in these orthotopic models to evaluate an antitumoral effect as well as a potential reduction of the edemas comparing the results in both models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2363. doi:1538-7445.AM2012-2363

Abstract B14: Characterization of an orthotopic glioblastoma in vivo mouse model with firefly luciferase expressing human U87 cells using in vivo bioluminescence imaging and magnetic resonance imaging (MRI).

Glioblastoma is the most common and most aggressive type of malignant brain tumors. The prognosis is poor, with a median survival time of several months only. Despite steady progress in treatment of glioblastomas this tumor entity is still not curable. Thus, there is an urgent need for the development of novel effective therapies, and for appropriate in vivo models to characterize them. Most often novel antiglioblastoma drugs are tested in subcutaneous xenograft mouse models using various human or murine glioblastoma cell lines. To generate more relevant, orthotopic brain cancer in vivo models, we stably transduced the human glioblastoma cell line U-87 with fire fly luciferase (U-87-Luci) suitable for in vivo bioluminescence imaging. To initiate orthotopic growth, nude mice were anesthetized and placed in a stereotactic fixation device. Through a burr hole cells were implanted intracranially using a Hamilton syringe. Once to twice a week we used bioluminescence imaging to continually monitor tumor growth in vivo. However, as magnetic resonance imaging (MRI) is the most relevant tool to monitor oncological therapy in glioblastomas clinically, we adapted this translational aspect and analysed the mice in parallel using a dedicated animal MRI System. MRI offers detailed information about growth characteristics, size, blood supply and interaction with the surrounding tissue of the tumor. To achieve this, we performed T2-weighted imaging, and diffusion weighted imaging (DWI) weekly to visualise the tumors as well as necrosis and edemas. To monitor the blood supply of the glioblastomas we performed dynamic contrast enhanced MR imaging (DCE-MRI). The use of both methods allowed independent and complementary characterization of the tumors. Approximately four weeks after implantation, exponential growth of the orthotopic tumors was observed, and another two weeks later necropsy was done. In vivo bioluminescence imaging revealed only a very dominant signal in the region of the brain where the primary tumor was located. To address potential metastases in more detail we analysed a set of different resected organs and tissues for in vitro luciferase activity followed by extensive histological examination. Currently, we test different known anti-glioblastoma agents in this orthotopic model to evaluate an antitumoral effect as well as a potential reduction of the edemas. Next, we plan to test carefully novel compounds in order to discover new generations of drugs suitable to help glioblastoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B14.

Abstract 3225: Cellular and in vivo models for the analyses of B-Raf and c-Src inhibitors

Cytosolic kinases c-Src and B-Raf represent proto-oncogenes of which mutant or overexpressed variants have been shown to cause cancer. Efforts are taken to develop inhibitors specifically addressing these kinases, some of which have already been approved such as Src inhibitor Dasatinib and BRaf inhibitor Sorafenib. In the current study we have generated cellular systems to allow for the analyses of Src and BRaf inhibitors in kinase specific cellular phosphorylation assays as well as in subcutaneous in vivo models. For both kinases we are introducing in vivo models allowing for regulatable kinase activity and thereby regulatable, kinase dependent tumor growth. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3225.