366 Targeting colorectal and pancreatic cancer stem cells with the LGR5 monoclonal antibody BNC101

118 Thursday 20 November 2014 365 POSTER Pim-1 kinase: Validated as a therapeutic cancer target for MYC-driven tumours O. Renner1 , Y. Cecilia1 , M.C. Rodriguez de Miguel1 , S. Peregrina1 , B. Garcia-Serelde1 , M.I. Albarran1 , A. Cebria1 , D. Cebrian1 , F. Ramos-Lima1 , A. Carnero1 , J. Pastor1 , C. Blanco-Aparicio1 . 1 Spanish National Cancer Research Centre (CNIO), Experimental Therapeutics Program, Madrid, Spain Provirus integration site for Moloney murine leukemia virus 1 (Pim-1) kinase belongs to a family of constitutively active serine/threonine kinases. The overexpression of Pim-1 can contribute to tumorigenesis, thereby establishing Pim-1 as a proto-oncogene. In B-cell lymphoma, colorectal cancer, pancreatic cancer and other tumours, the over expression of Pim-1 is linked to poor prognosis. Proviral integration experiments suggested the cooperation between Pim-1 and the protooncogene c-myc that was subsequently confirmed by double transgenic mice. Pim-1 acts synergistically with the transcription factor c-myc by phosphorylation and by interaction with c-myc on the chromatin level, leading to elevated trancription of c-myc target genes and cell proliferation. Pim-1 also promotes cell cycle progression. Pim-1-mediated phosphorylation of p27 leads to its binding to 14−3−3 protein, its nuclear export and proteasome-dependent degradation. The aim of our study was to analyze the effect of kinase-inhibition of Pim-1 on tumour growth in a mouse model of haematological malignancy. To this end, we constructed a conditional knock-in mouse model in which a single amino acid exchange can be induced by expression of the Cre-recombinase, leading to expression of an inactive form of Pim-1 kinase. These Pim-1 kinase-dead (KD) mice do not display phenotypic abnormalities or reduced live span even after homozygous exchange by CMV-Cre expression of the wild type form of Pim-1 against the mutated form. As expected, elevated levels of the cell cycle inhibitor p27 can be observed in the organs of Pim-1 kinase-dead mice by immunohistochemistry (IHC). In order to study the effect of the Pim-1 kinase inhibition on tumour development driven by c-myc, we crossed the Pim-1 KD mice with the Em-cmyc transgenic mouse line, which expresses high levels of the oncoprotein c-myc leading to the development of a rapidly growing B-cell lymphoma that resembles the human Burkitt-lymphoma. Even the heterozygous switch to the kinase-dead form of Pim-1 doubles the survival of the Em-c-myc mice; the homozygous switch brings survival back close to normal. On the molecular level, we observed elevated p27-levels in tumours of hetero- and homozygous Pim-1 kinase-dead mice carrying the Em-c-myc transgene. The tumours of these mice also displayed areas with reduced levels of c-myc (Western blot and IHC). Based upon these results, we set-up an allogenic Em-c-myc mouse model that allowed us to study the effect of kinase inhibitors in an efficient and standardized way. We can show that novel, selective and potent Pim-1 kinase inhibitors that have been developed in our department are able to efficiently inhibit c-myc-driven tumour growth, as proposed by the genetic ablation of Pim-1 kinase activity. In summary, the presented study proofs inhibition of Pim-1 kinase activity to be a promising strategy to inhibit cancer growth driven by c-myc, one of the most frequently deregulated oncogenes in human cancer. 366 POSTER Targeting colorectal and pancreatic cancer stem cells with the LGR5 monoclonal antibody BNC101 P. Chu1 , F. Shojaei1 , K. Smith1 , J. Norton1 , C. Walsh1 , J. Iglesias1 , C. Reyes1 . 1 Bionomics Inc, San Diego CA, USA BNC101 is a monoclonal antibody (mAb) targeting LGR5 currently undergoing IND-enabling studies in preparation for Phase I clinical studies in 2014. LGR5 is a validated cancer stem cell (CSC) receptor overexpressed in colorectal cancer (CRC), pancreatic cancer and most other solid tumors. Loss and gain-of-function studies indicate that LGR5 is a functional cancer receptor involved in the growth and survival of colon cancer cells in vitro and in vivo. Sorted LGR5+ primary CRC are highly tumorigenic compared to LGR5− cells in limiting dilution in vivo xenograft studies. Using Bionomics’ CSC Rx Discovery™ platform, a panel of high affinity LGR5 mAbs were generated and screened for in vitro and in vivo activity against CSCs from patient-derived CRC tumors. LGR5 mAb therapeutic drug candidates were first screened and identified based on their ability to inhibit the formation of CRC CSC colonies in vitro. Top LGR5 mAb candidates were then tested as single agents and in combination with standard of care (SOC) chemotherapy in primary xenograft efficacy studies. Tumors from these studies were also harvested and re-implanted in limiting dilution assays (LDA) to assess in vivo anti-CSC activity of antiLGR5 mAbs. A number of functional LGR5 mAbs with significant antitumor and anti-CSC activity were identified through this process. A lead

Poster Session – Molecular Targeted Agents I BNC101 mAb clone was selected for clinical development based on its ability to inhibit CSC growth and key signaling pathways from multiple patient tumors both in vitro and in vivo. In LDA studies using two different CRC patient tumors, 5/8 (63%) and 6/8 (75%) mice implanted with serially diluted cells from BNC101 treated tumors remained tumor free, compared to 1/8 (13%) and 2/8 (25%) in the control groups. Combination with SOC was shown to further improve BNC101 activity in one CRC model, where 8/8 mice re-implanted for LDA remained tumor-free in a 6 month followup. BNC101 was also found to be highly active as a single agent and in combination with SOC in multiple pancreatic models and the MDA-MB231 triple-negative breast cancer (TNBC) xenograft model. In one primary pancreatic model, BNC101 combined with SOC completely eradicated 3/7 established tumors, compared to 0/7 tumors eradicated with control SOC alone. Additional in vivo translational data in CRC, pancreatic and TNBC supporting our hypothesis that BNC101 targeting of CSCs will significantly improve survival and increase duration of response in cancer patients will be presented. 367 POSTER Induction of apoptosis and inhibition of angiogenesis by novel fusion protein − AD-O54.9 as a new preclinical strategy in cancer treatment P. Rozga1 , B. Zerek1 , A. Pieczykolan1 , M. Galazka1 , K. Bukato1 , S. Pawlak1 , M. Szymanik1 , A. Jaworski1 , M. Teska-Kaminska1 , K. Poleszak1 , A. Grochot-Przeczek2 , W. Strozek1 , J. Pieczykolan1 . 1 Adamed, Drug Discovery, Warsaw, Poland; 2 Jagiellonian University, Department of Medical Biotechnology, Krakow, Poland Background: For almost two decades tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) has been under extensive development as a potential therapeutic agent. TRAIL/Apo2L is a member of the TNF superfamily with unique ability to induce apoptosis in cancer cells while remaining neutral to normal cells. Tumor growth is tightly related to new blood vessel formation and tissue remodeling. Platelet derived growth factor (PDGF) is important in vascular physiological and pathological development. Inhibition of PDGF pathway blocks angiogenesis what results in reduction of tumor growth. We proposed a novel fusion protein (AD-O54.9) with dualistic proapoptotic and antiangiogenic activity. Our molecule consists of the soluble recombinant TRAIL/Apo2L variant linked with an effector peptide sequence and an activation motif recognized by tumor-specific proteases (MMPs, uPa) between. The effector peptide is composed of 19-amino acid fragment derived from human PDGF, which binds the PDGF receptors competitively to the natural ligand while being itself devoid of activity. As a consequence, angiogenic activity of PDGF is blocked, stimulation of new blood vessels formation does not occur and finally tumor growth is inhibited. Materials and Methods: AD-O54.9 protein was expressed in E. coli, using pET expression system, and purified by IEX chromatography. The biophysical properties were verified by circular dichroism (CD) and interactions with receptors were analyzed using surface plasmon resonance (SPR). Cytotoxic activity of AD-O54.9 was examined using a propidium iodide assay and its antiangiogenic activity was evaluated by HUVEC tube formation and ring aortic assays. Safety was tested on human primary hepatocytes. The proapoptotic and antiproliferative activity was tested using molecular biology and flow cytometry methods. In vivo potential was examined on mice xenograft models of human cancers. Results: The molecule showed in vitro specific cytotoxic effect on various primary cancer cell lines at IC50 below 0.01 ng/ml with no activity against human hepatocytes. This protein efficiently interacted with TRAIL and PDGF receptors. We demonstrated that AD-O54.9 is a very potent apoptosis inducer and inhibitor of angiogenesis. This fusion protein showed superior efficacy displaying significant tumor volume regression compared with TRAIL/Apo2L and standard chemotherapeutic agents. Conclusions: The obtained results confirm that we developed a very promising fusion molecule with a high potential of anticancer activity that could be considered as a novel therapeutic agent. 368 POSTER NP137, the first humanized monoclonal antibody directed against netrin-1, exhibits antitumor activity by inducing dependence receptors-mediated cell death J.G. Delcros1 , B. Ducarouge1 , R. Abes2 , D. Goldschneider1 , B. Gibert1 , J.G. Blachier2 , D. Neves2 , P. Mehlen1 , A. Bernet2 , S. Depil2 . 1 Cancer Research Center of Lyon, Dependence receptors cancer and development, Lyon Cedex 08, France; 2 Netris Pharma, Lyon Cedex 08, France Background: Some receptors are active in the absence of ligand and actively trigger cell death through apoptosis. These receptors are called