Kerstin Klingner

Effective Eradication of Glioblastoma Stem Cells by Local Application of an AC133/CD133-Specific T-cell–Engaging Antibody and CD8 T Cells

Cancer stem cells (CSC) drive tumorigenesis and contribute to genotoxic therapy resistance, diffuse infiltrative invasion, and immunosuppression, which are key factors for the incurability of glioblastoma multiforme (GBM). The AC133 epitope of CD133 is an important CSC marker for GBM and other tumor entities. Here, we report the development and preclinical evaluation of a recombinant AC133×CD3 bispecific antibody (bsAb) that redirects human polyclonal T cells to AC133(+) GBM stem cells (GBM-SC), inducing their strong targeted lysis. This novel bsAb prevented the outgrowth of AC133-positive subcutaneous GBM xenografts. Moreover, upon intracerebral infusion along with the local application of human CD8(+) T cells, it exhibited potent activity in prophylactic and treatment models of orthotopic GBM-SC-derived invasive brain tumors. In contrast, normal hematopoietic stem cells, some of which are AC133-positive, were virtually unaffected at bsAb concentrations effective against GBM-SCs and retained their colony-forming abilities. In conclusion, our data demonstrate the high activity of this new bsAb against patient-derived AC133-positive GBM-SCs in models of local therapy of highly invasive GBM.

Intratibial Injection of Human Multiple Myeloma Cells in NOD/SCID IL-2Rγ(Null) Mice Mimics Human Myeloma and Serves as a Valuable Tool for the Development of Anticancer Strategies

Background We systematically analyzed multiple myeloma (MM) cell lines and patient bone marrow cells for their engraftment capacity in immunodeficient mice and validated the response of the resulting xenografts to antimyeloma agents. Design and Methods Using flow cytometry and near infrared fluorescence in-vivo-imaging, growth kinetics of MM cell lines L363 and RPMI8226 and patient bone marrow cells were investigated with use of a murine subcutaneous bone implant, intratibial and intravenous approach in NOD/SCID, NOD/SCID treated with CD122 antibody and NOD/SCID IL-2Rγ(null) mice (NSG). Results Myeloma growth was significantly increased in the absence of natural killer cell activity (NSG or αCD122-treated NOD/SCID). Comparison of NSG and αCD122-treated NOD/SCID revealed enhanced growth kinetics in the former, especially with respect to metastatic tumor sites which were exclusively observed therein. In NSG, MM cells were more tumorigenic when injected intratibially than intravenously. In NOD/SCID in contrast, the use of juvenile long bone implants was superior to intratibial or intravenous cancer cell injection. Using the intratibial NSG model, mice developed typical disease symptoms exclusively when implanted with human MM cell lines or patient-derived bone marrow cells, but not with healthy bone marrow cells nor in mock-injected animals. Bortezomib and dexamethasone delayed myeloma progression in L363- as well as patient-derived MM cell bearing NSG. Antitumor activity could be quantified via flow cytometry and in vivo imaging analyses. Conclusions Our results suggest that the intratibial NSG MM model mimics the clinical situation of the disseminated disease and serves as a valuable tool in the development of novel anticancer strategies.

Abstract 1026: Novel Tie2 inhibitor with in vivo efficacy in disseminated hematological tumor models in mice

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The receptor tyrosine kinase Tie2 is predominantly expressed in the endothelium but has also been identified on primitive hematopoietic stem cells, monocyte and macrophage subclasses, as well as on glioma or hematological tumor cells. Based on its expression in many patient-derived leukemic blasts inhibition of the Tie2 pathway may provide an attractive opportunity for therapeutic intervention in leukemias. In this study we report the pharmacological profile of a novel, highly potent and orally available Tie2 inhibitor (BAY-Tie2). The discovery and design process leading to BAY-Tie2 was performed with the goal of sparing other angiogenic RTKs, such as VEGFRs, FGFRs or PDGFRs. BAY-Tie2 is based on a novel imidazopyrazole core, combined with a SF5-substituted phenyl ring that fills the deep DFG-out pocket. BAY-Tie2 binds to Tie2 with a Kd value of 1.6 nM and is selective against VEGFR2 (Kd of 1600 nM), FGFR1 (<30% inhibition at 1 µM), FGFR2/3/4 (<10% inhibition at 1 µM) and PDGFRα/β (<30% inhibition at 100 nM). BAY-Tie2 potently inhibits Tie2 autophosphorylation in recombinant CHO-Tie2 and primary human umbilical vein endothelial cells (HUVEC) with IC50 values of 0.7 and 1.3 nM. Consistently, BAY-Tie2 was shown to inhibit Tie2 phosphorylation in vivo by analyzing angiopoietin-1 induced Tie2 phosphorylation status in extracts of murine lungs from BAY-Tie2-treated mice. In subcutaneous xenograft models of highly angiogenic tumors, BAY-Tie2 reduced tumor growth and showed evidence for potential combination benefit with anti-VEGF therapy. In order to explore the potential of a Tie2 inhibitor beyond affecting angiogenesis, we established disseminated leukemia models, using Tie2-expressing cell lines, such as the CML cell lines MEG-01 and EM-2. Both cell lines engrafted predominantly in bone marrow and spleen. Treatment started 3 days after i.v. cell implantation with either BAY-Tie2 or cytarabine and was well tolerated. Efficacy was monitored by a) inhibition of disease progression, b) weekly fluorescence-based in vivo imaging (IVI) using an Alexa750-labeled anti-human CD33 antibody, and c) q-RT-PCR specific for BCR-ABL and hCD45 in murine peripheral blood. BAY-Tie2 inhibited disease progression comparable to cytarabine. Tumor load measured by IVI was reduced in BAY-Tie2 treated groups by 45% in the MEG-01 and by 65% in the EM-2 model compared with the untreated control, very similar to the cytotoxic treatment with cytarabine. Quantitative RT-PCR on peripheral blood revealed that BAY-Tie2 and cytarabine delayed the appearance of circulating tumor cells in both CML models. These data demonstrate that BAY-Tie2 is an orally active Tie2 inhibitor that may have therapeutic benefit not only in angiogenic tumors but also in hematological, Tie2-expressing malignancies. Citation Format: Sylvia Gruenewald, Julia Schueler, Michael Haerter, Frank Suessmeier, Kerstin Klingner, Ulf Boemer, Stefan Kaulfuss, Alexander Walter, Mario Lobell, Ingo V. Hartung, Bernd Buchmann, Dieter Heldmann, Holger Hess-Stumpp, Karl Ziegelbauer. Novel Tie2 inhibitor with in vivo efficacy in disseminated hematological tumor models in mice. [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 1026. doi:10.1158/1538-7445.AM2014-1026

Patient derived renal cell carcinoma xenografts exhibit distinct sensitivity patterns in response to antiangiogenic therapy and constitute a suitable tool for biomarker development

Systemic treatment is necessary for one third of patients with renal cell carcinoma. No valid biomarker is currently available to tailor personalized therapy. In this study we established a representative panel of patient derived xenograft (PDX) mouse models from patients with renal cell carcinomas and determined serum levels of high mobility group B1 (HMGB1) protein under treatment with sunitinib, pazopanib, sorafenib, axitinib, temsirolimus and bevacizumab. Serum HMGB1 levels were significantly higher in a subset of the PDX collection, which exhibited slower tumor growth during subsequent passages than tumors with low HMGB1 serum levels. Pre-treatment PDX serum HMGB1 levels also correlated with response to systemic treatment: PDX models with high HMGB1 levels predicted response to bevacizumab. Taken together, we provide for the first time evidence that the damage associated molecular pattern biomarker HMGB1 can predict response to systemic treatment with bevacizumab. Our data support the future evaluation of HMGB1 as a predictive biomarker for bevacizumab sensitivity in patients with renal cell carcinoma.

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays(1). Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research (2-4). Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors(4) or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable. Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis (5). To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Abstract 590: Co-injection of human monocytes improves the in vivo antitumoral activity of bevacizumab in two NSCLC PDX models

Nowadays, an increasing number of monoclonal antibodies (mAbs) that specifically target malignant cells or interfere with different compartments of the tumor microenvironment are available for cancer therapy. They take effect via different modes of action including the initiation of a tumor-targeting immune response. Preclinical platforms such as PDX models have to be improved to better recapitulate all possible modes of action for mAbs as well as other immune-modulating agents. In the current study, we evaluated the antitumoral activity of Bevacizumab in two NSCLC PDX growing subcutaneously in NMRI nu/nu mice with and without co-injection of human monocytes. Two NSCLC PDX, LXFA 2478 and LXFA 677, were subcutaneously implanted into 4-6 weeks old female NMRI nu/nu mice (Harlan, Denmark). Tumor models were chosen based on their VEGFA expression level. Additionally, LXFA 2478 and LXFA 677 show high expression of TLR2 and CD14 respectively, two factors known to be involved in monocyte attraction. When median tumor size reached 150 - 300 mm3, mice were equally distributed to treatment groups (n = 4/group). Animals were treated once weekly for 7 cycles with a) control vehicle b) control vehicle + 5×106 human monocytes c) Bevacizumab at 40 mg/kg/d and d) Bevacizumab + 5×106 human monocytes. Tumor volume was determined twice weekly by caliper measurement. At the end of the study, tumor and lymphatic organs of the animals were harvested and subsequent IHC analysis for human CD14, CD68 and CD163 was performed. In both investigated tumor models, Bevacizumab showed moderate antitumoral activity with a maximal tumor load reduction of 71% (LXFA 2478) and 84% (LXFA677) as compared to untreated controls on days 39 and 35. The co-injection of human monocytes markedly enhanced the therapeutic effect of Bevacizumab in both NSCLC PDX: maximal tumor load reduction was 89% in LXFA 2478 and 95% in LXFA 677 in combination groups. The injection of monocytes alone did not affect tumor growth as compared to untreated control. As predicted by the high VEGFA expression, NSCLC PDX showed high sensitivity against Bevacizumab. This antitumoral activity was increased by 18% and 11% for LXFA 2478 and LXFA 677, respectively, through the additional injection of monocytes. Monocyte recruiting factors (namely CD14 and TLR2) likely contribute to the mechanism of action. Monocytes and macrophages have been reported to induce antibody-dependent cytotoxicity and phagocytosis of tumor cells in the presence of IgG anti-tumor mAbs, like Bevacizumab. Our results confirm these observations in a PDX based NSCLC in vivo model. Therefore, the study highlights the suitability of PDX for immuno-oncology approaches by supplementation of the murine host with human immune cells. This advanced PDX approach will lead to more predictive preclinical data for innovative mAb′s and other compounds acting via the activation of monocytes and related immune cells. Citation Format: Cordula Tschuch, Kerstin Klingner, Anne Lohr, Yana Raeva, Teppo Haapaniemi, Eva Oswald, Julia B. Schuler. Co-injection of human monocytes improves the in vivo antitumoral activity of bevacizumab in two NSCLC PDX models. [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 590.

Abstract A10: Establishment and characterization of a patient-derived non-small cell lung cancer mouse model of acquired resistance towards anti-EGFR treatment

Non-small cell lung cancer (NSCLC) is the largest subgroup of lung cancer, occurring at a frequency of over 80% of lung cancer cases. In up to 30% of NSCLC patients the oncogenic driver of tumor growth is a constitutively activated EGF receptor (EGFR), which plays a critical role in regulating multiple cellular processes, including proliferation, survival and apoptosis. Although these patients gain great benefit from treatment with EGFR tyrosine kinase inhibitors (TKI, e.g. erlotinib or gefitinib), development of resistance is inevitable. To model the emergence of drug resistance, an EGFR driven, gefitinib sensitive, patient-derived xenograft (PDX) NSCLC model was treated continuously with gefitinib in immunocompromised mice. The dose of daily treatment was adjusted according to tumor growth over a period of up to 91 days. In a first phase, dosing was high (40-50 mg/kg) to eradicate EGFR TKI sensitive cells. At the time point of maximal antitumoral activity dosing was reduced to 20-30 mg/kg ( = low dose) to preserve selection pressure. Between 69 and 91 days after dosing was initiated, drug-resistant tumors emerged in 4 out of 10 mice under high dose treatment. Resistant tumor fragments, which were re-implanted into a new cohort of mice and continuously treated with gefitinib, kept resistance also under high dose treatment. A comprehensive analysis using Western blot (WB), qPCR and sequencing was performed to identify the reason for resistance. In WB analysis we could show that signalling through EGFR was completely abrogated in all four resistant tumor sublines. Neither secondary mutations in EGFR (ex19-21) or KRAS (ex 1+2) could be detected, nor was the expression of cMET, AXL, HGF, PTEN or HER3 significantly increased in resistant tumors as shown by sequencing and qPCR respectively. However a more comprehensive WB analysis revealed several genes being activated in resistant compared to primary tumors. Depending on the subline, phospho-(p)-cMET, p-AKT, AXL & p-AXL, p-cRAF, p-MEK, p-ERK as well as HER3, IGF-R and ALK were up-regulated in resistant tumors. Based on these data we determined that signalling pathways such as the (RAS)-cRAF-MEK-ERK signalling cascade or enhanced cMET/AKT signalling were activated in drug-resistant tumors. These signalling pathways are known to be alternative pathways for EGFR signalling also in patients with acquired resistance indicating the clinical relevance of these models. To shed more light into the mechanism of resistance, whole exome sequencing analyses of the four resistant sublines as well as the original tumor model are underway. In summary, we have developed four NSCLC tumor sublines each harbouring a different mechanism of resistance to EGFR TKI treatment, modelling the emergence of drug-resistant NSCLC in patients. Herewith a strong preclinical tool for the development of innovative compounds targeting acquired EGFR resistance is now available. Citation Format: Cordula Tschuch, Kerstin Klingner, Dorothee Lehnhard, Anne Lohr, Yana Raeva, Anne-Lise Peille, Eva Oswald, Julia B. Schuler. Establishment and characterization of a patient-derived non-small cell lung cancer mouse model of acquired resistance towards anti-EGFR treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A10.

Next-generation sequencing of human tumor xenografts is significantly improved by prior depletion of mouse cell

Human tumor xenografts represent the gold standard method for many research areas, including drug discovery, cancer stem cell biology, and metastasis prediction. When compared to in vitro cell culture models, human tumor xenografts show a higher validity for most assays1. During the growth phase in vivo, xenografted tissue is vascularized and infiltrated by cells of murine origin. The level of infiltration is highly dependent on multiple factors like tumor subtype, growth rate, and region of transplantation. However, even when these factors are kept constant, the amount and composition of infiltrating mouse cells is highly variable. Due to this, molecular downstream analyses such as microarray-based expression profiling are biased by cross-hybridization of mouse-derived molecules to human probes. In addition, a reduction of sensitivity caused by measuring undesired mouse signals during nextgeneration sequencing analysis can be expected. To overcome these limitations, we developed a fast and easy method allowing for the effective depletion of all cells of mouse origin by using automated tissue dissociation and magnetic cell sorting (MACS® Technology). We performed whole exome sequencing (WES) of bulk human tumor xenografts from lung, bladder, and kidney cancer, and compared the results to samples depleted of mouse cells.

Abstract 5023: NSCLC PDX model for the evaluation of immuno-oncological treatment strategies

Patient-derived tumor xenografts (PDX) have played a major role in the development of new cancer therapies and their strengths and weaknesses have gradually been elucidated. One major drawback of PDX is the lack of an immunological competent host. To overcome this hurdle we supplemented NSG/NOG mice with human hematopoietic stem cells (HSC) and subsequently examined growth characteristics of the non-small cell lung cancer (NSCLC) PDX model LXFA 923 in these mice. In parallel we monitored the presence of human and murine immune cells in different organs of the mouse. HSC (2×106) cells were isolated from healthy donors and injected intravenously into sub-lethally irradiated NSG or NOG mice (n = 43 mice in 3 ind. exp.). After 8 weeks LXFA 923 was transplanted subcutaneously (s.c.) into the pretreated mice. Murine peripheral blood was examined by flow cytometry for common murine and human markers expressed on immune cells (hCD14, mCD14, hCD3, mCD3, hCD56, mCD56, hCD19) once weekly. At the end of the experiment tumors and organs were analyzed for human cancer (CD44, CD133, CDCP1, CD166, CD24) and immune cell markers (hCD14, hCD3, hCD56, hCD19) by flow cytometry. Tumors and organs were additionally histologically and immunohistochemically examined. Growth of the implanted tumors was monitored by caliper measurement. Mice bearing only the subcutaneous PDX or the HSC served as control groups. Stable engraftment of human immune cells in immune-compromized mice was successfully achieved. Human immune cells expressing T-, B-, NK- and stem cell markers could be detected in different compartments (bone marrow, peripheral blood and spleen) of the tumor-bearing as well as non-tumor bearing mice. Furthermore, infiltrates of human monocytes (CD14+) as well as T cells (CD3+) could be detected in s.c. implanted tumor tissue. Implantation of LXFA 923 did not influence the proliferation of human immune cells in recipient mice. Growth behavior of the s.c. implanted PDX was not affected by the engraftment of HSC in the murine host. The histological architecture of LXFA 923 was similar when implanted s.c. in humanized or immunodeficient mice and it still closely resembles the patient donor material. In conclusion, our investigations validate the analysis of PDX in mice engrafted with human immune cells, as it enables the interaction of tumor cells with human immune cells as well as with murine stroma to be investigated. This preclinical PDX based in vivo platform provides a further step to support the development of new drugs targeting the host immune response. Citation Format: Eva Oswald, Kerstin Klingner, Dorothee Lenhard, Gabriele Niedermann, Julia B. Schuler. NSCLC PDX model for the evaluation of immuno-oncological treatment strategies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5023. doi:10.1158/1538-7445.AM2015-5023

Abstract 1455: Next generation sequencing of human tumor xenografts is significantly improved by prior depletion of mouse cells

Human tumor xenografts represent the gold standard method for many research areas, including drug discovery, cancer stem cell biology, and metastasis prediction. When compared to in vitro cell culture models, human tumor xenografts show a higher validity for most assays1. During the growth phase in vivo, xenografted tissue is vascularized and infiltrated by cells of murine origin. The level of infiltration is highly dependent on multiple factors like tumor subtype, growth rate, and region of transplantation. However, even when these factors are kept constant, the amount and composition of infiltrating mouse cells is highly variable. Due to this, molecular downstream analyses such as microarray-based expression profiling are biased by cross-hybridization of mouse-derived molecules to human probes. In addition, a reduction of sensitivity caused by measuring undesired mouse signals during nextgeneration sequencing analysis can be expected. To overcome these limitations, we developed a fast and easy method allowing for the effective depletion of all cells of mouse origin by using automated tissue dissociation and magnetic cell sorting (MACS® Technology). We performed whole exome sequencing (WES) of bulk human tumor xenografts from lung, bladder, and kidney cancer, and compared the results to samples depleted of mouse cells.