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In-vitro and in vivo preclinical evaluation of a 5T4 specific antibody–drug conjugate for treating ovarian cancer
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 645 Investigation of the anti-cancer potential of natural products and their synthetic analogues B. Adegbesan1 , C. Demonacos1 . 1 University of Manchester, Manchester Pharmacy School, Manchester, United Kingdom Introduction: NRH quinone-oxidoreductase 2 is one of the two members of the quinone-oxidoreductase gene family; it is an antioxidative enzyme involved in the two-electron reduction of quinones directly to hydroquinones. The biological function of NQO2 has not yet been elucidated. NQO2 is overexpressed in some types of cancer therefore inhibiting its enzymatic activity could potentially be used for the treatment of such types of cancer. The mammalian sirtuins are a class of proteins possessing either monoribosyltransferase or deacylase activity and have been reported to be involved in cellular stress resistance. Many natural agents and synthetic analogues targeting both NQO2 and sirtuins have been used as experimental drugs in cancer prevention and treatment. Resveratrol (3, 4 5-trihydroxy-transstilbene), a member of polyphenols has been shown to inhibit the enzymatic activity of NQO2 and activate sirtuins. Sirtinol, a cell-permeable inhibitor of sirtuins has been reported to inhibit the growth of cancer cells. This study is designed to explore pathways that could be used to design novel cancer therapeutics based on the biological effects mediated by NQO2 and Sirt-1 using resveratrol, sirtinol and other synthetic analogues in breast cancer cells. Materials and Methods: The interrelation between Sirt-1 and NQO2 was investigated by probing for p53 and E2F1 protein levels in MDA-MB-231, MDAMB-468, MCF7 and T47D breast cancer cell lines using western blot analysis; the effect of these activators and inhibitors on cell cycle progression and ROS generation were determined by FACs; the potential NQO2 and/or Sirt-1 mediated regulation of p53 and E2F1 transcriptional activities was investigated using Cyclin-E luciferase reporter assay. Results and Discussion: The results from this study indicate that NQO2 and Sirt-1 potentially regulate p53 and E2F1 protein levels in a cell type dependent manner. Also, treatment with resveratrol, sirtinol and other synthetic compounds in these cells affect p53 and E2F1 levels differently suggesting that they may serve as important tools to dissect the synergistic and antagonistic effects of Sirt-1 and NQO2 on these cell cycle and apoptosis main regulators. Cell cycle analyses reveal that resveratrol, sirtinol and other synthetic analogues cause arrest of cell cycle at either the G1 or S phase. ROS analyses indicate that these compounds affect the level of intracellular ROS differentially. Cyclin-E Luciferase assays reveal that the transcriptional activities of E2F1 and p53 are also differentially regulated by the different treatments in the cell lines. Conclusion: Resveratrol, sirtinol and other synthetic analogues cause arrest of cell cycle at either the G1 or S phase in breast cancer cells an effect that make them suitable to be explored as therapeutic tools in breast cancer. No conflict of interest. 646 AAV-shRNA vectors as an alternative therapy for human basal-like breast cancer C. Pinto1,2 , A.S. Ribeiro3 , G. Silva1 , M. Oliveira3 , A.S. Coroadinha1,2 , C. Peixoto1,2 , A. Barbas1 , C. Brito1,2 , J. Paredes3 , P.M. Alves1,2 . 1 IBET´ Instituto de Biologia Experimental e Tecnologica, Animal Cell Technology ´ ´ Unit, Oeiras, Portugal, 2 Instituto de Tecnologia Qu´ımica e Biologica Antonio Xavier- Universidade Nova de Lisboa, Animal Cell Technology Unit, Oeiras, Portugal, 3 IPATIMUP, Epithelial Interactions in Cancer, Porto, Portugal Background: Basal-like breast cancer (BBC) is mainly comprised by triple-negative breast cancers (TNBC) which display a highly aggressive and metastatic phenotype. These tumors rapidly acquire resistance to chemotherapy, are refractory to endocrine therapy and HER2 inhibitors, and have no targeted therapeutic options. As a consequence, they present a poor clinical outcome. Therefore, there is a need to find alternative therapies against this type of tumors. In this study, we aimed to develop a novel gene therapybased treatment for BBC based on viral vector-mediated delivery of shRNA targeting BBC cell survival genes, such as MCL1, PSMA2 and PSMB4. Materials and Methods: pAAV2-shRNA plasmids were generated by cloning shRNA sequences targeting previously identified BBC dependency genes, or validated scramble control shRNA sequences, together with GFP reporter gene between AAV2 inverted terminal repeats in an AAV plasmid backbone. AAV2-shRNA vectors were produced by 2-plasmid co-transfection of HEK293T cells with pAAV2-shRNA plasmids and the pDG plasmid, and purified by affinity chromatography. Target gene knockdown efficiency and functional effects of the different AAV2-shRNA vectors in BBC cells were evaluated by qRT-PCR, Presto Blue analysis and flow cytometry. The anti-tumorigenic effect of AAV2PSMA2sh vector was evaluated on mouse orthotopic BBC cell xenografts. Results: AAV2-shRNA vectors against several BBC genes and control scramble vectors (negative controls) were produced with high titers and high purity. Transduction of two BBC cell lines (MDA-MB-468 and HCC1954) with AAV2-PSMA2sh decreased the expression of its target gene by 80%, as compared to control cells transduced with AAV2 vector encoding a scramble shRNA (negative control). In MDA-MB-468 cells, knockdown of PSMA2 gene
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was associated with significant decrease in cell viability and 2 fold increase in the percentage of apoptotic cells. In vivo studies to assess the anti-tumorigenic effect of AAV2-PSMA2sh in mouse orthotopic BBC cell xenografts showed that intratumoral injections with this vector caused a significant decrease in tumor growth, when compared to negative scramble shRNA control vector or PBS injected mice. No major macroscopic alterations were observed in the liver of mice treated with AAV-shRNA vectors, as compared to PBS control. Assays are currently in progress to further characterize the tumors, e.g. proliferation and apoptotic status. Conclusions: These results indicate that AAV2-PSMA2sh vector decreases BBC tumorigenesis and suggest that PSMA2 gene may represent a potential therapeutic target for BBC therapy. No conflict of interest. 647 In-vitro and in vivo preclinical evaluation of a 5T4 specific antibody–drug conjugate for treating ovarian cancer Y.L. Wan1 , P. Sapra2 , D. Gilham3 , H.C. Kitchener1 , P.L. Stern1 . 1 University of Manchester, Gynaecological Oncology Research Group- Institute of Cancer Sciences, Manchester, United Kingdom, 2 Pfizer Worldwide Research & Development, Bioconjugates Discovery & Development- Oncology Research Unit, Pearl River, NY, USA, 3 University of Manchester, Clinical and Experimental Immunotherapy Group- Institute of Cancer Sciences, Manchester, United Kingdom Background: The oncofoetal glycoprotein, 5T4, is strongly expressed by many different cancers but has little expression in normal adult tissues. Its expression is associated with advanced disease and poorer clinical outcome in a number of cancer types, including ovarian cancer. 5T4 is mechanistically associated with the directional movement of cells through the regulation of epithelial mesenchymal transition, facilitation of chemotaxis and modulation of Wnt signaling. The tumour selective expression of 5T4 has prompted the development of several immunotherapies; including a 5T4 antibody– drug conjugate (A1mcMMAF). The latter is a 5T4 humanized monoclonal antibody (A1) linked by sulfydryl-based conjugation to deliver a microtubuledisrupting agent, monomethyl auristan F (MMAF) via a maleimidocaproyl linker. A1mcMMAF has shown potent in vivo activity in a variety of tumour models, with induction of long term regression after the last dose. Here we test activity versus ovarian cancer cells. Methods: In vitro IC50 values of 5T4-ADC in SKOV3 cells expressing 5T4 and 5T4 knock out cells (SKOV3-5T4KO) were determined using MTS assays. Apoptosis and viability were assessed by Apotox-Glo assay. Anti-tumor activity of A1mcMMAF with or without carboplatin chemotherapy against SKOV3luciferase xenografts in NSG mice models was assessed using bioluminescent imaging. Results: A1mcMMAF selectively killed SKOV3 cells with an IC50 20 fold lower than that seen in SKOV3-5T4KO cells. Within the selective range, A1mcMMAF caused apoptosis in SKOV3 but not SKOV3-5T4KO cells. A1mcMMAF (5 mg/kg IP) treatment of SKOV3 xenografts reduced tumour load, delayed growth and significantly increased survival compared to untreated animals. A combination of A1mcMMAF with optimized dosing of carboplatin showed no toxicity and a greater reduction in tumour load and improved survival compared to A1mcMMAF monotherapy. A1mcMMAF plus carboplatin (25 mg/kg IP) showed a median survival increase of 80% compared to carboplatin alone. Conclusions: A1mcMMAF is selective for cells expressing 5T4 and shows potent anti-tumor activity in SKOV3 xenograft models. These data support the use of A1mcMMAF in ovarian cancer treatment potentially in combination with carboplatin. Conflict of interest: Other Substantive Relationships: A1McMMAF was developed by Pfizer Inc and kindly donated for use in this project which was funded by the Wellcome Trust. Puja Sapra is an employee of Pfizer Inc. and owns company’s stock options and/or units. Peter Stern has received honoraria from Pfizer Inc. 648 Membrane Hsp70 as a biomarker for aggressive prostate cancer and therapeutic target G.A. Foulds1 , N. Dunning-Foreman1 , S. Stangl2 , M. Gehrmann2 , J. Vadakekolathu1 , D. Boocock1 , R. Rees1 , G. Multhoff2 , A.G. Pockley1 . 1 Nottingham Trent University, John van Geest Cancer Research Centre, ¨ Munchen, Nottingham, United Kingdom, 2 Technische Universitat ¨ Department of Radiation Oncology, Munich, Germany Introduction: Professor Gabriele Multhoff first identified the selective expression of a membrane form of Hsp70 (memHsp70) on the plasma membrane of tumour cells (but not normal tissue) using a unique monoclonal antibody (mAb, cmHsp70.1, multimmune GmbH). The expression of memHsp70 has since been shown to associate with aggressive disease (e.g. metastasis) and an unfavourable prognosis and poorer survival in patients with cancer. An ongoing screening program of solid tumours at the Technische Universitat ¨ Munchen ¨ is revealing that more than 50% of all tumours express memHsp70.
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was associated with significant decrease in cell viability and 2 fold increase in the percentage of apoptotic cells. In vivo studies to assess the anti-tumorigenic effect of AAV2-PSMA2sh in mouse orthotopic BBC cell xenografts showed that intratumoral injections with this vector caused a significant decrease in tumor growth, when compared to negative scramble shRNA control vector or PBS injected mice. No major macroscopic alterations were observed in the liver of mice treated with AAV-shRNA vectors, as compared to PBS control. Assays are currently in progress to further characterize the tumors, e.g. proliferation and apoptotic status. Conclusions: These results indicate that AAV2-PSMA2sh vector decreases BBC tumorigenesis and suggest that PSMA2 gene may represent a potential therapeutic target for BBC therapy. No conflict of interest. 647 In-vitro and in vivo preclinical evaluation of a 5T4 specific antibody–drug conjugate for treating ovarian cancer Y.L. Wan1 , P. Sapra2 , D. Gilham3 , H.C. Kitchener1 , P.L. Stern1 . 1 University of Manchester, Gynaecological Oncology Research Group- Institute of Cancer Sciences, Manchester, United Kingdom, 2 Pfizer Worldwide Research & Development, Bioconjugates Discovery & Development- Oncology Research Unit, Pearl River, NY, USA, 3 University of Manchester, Clinical and Experimental Immunotherapy Group- Institute of Cancer Sciences, Manchester, United Kingdom Background: The oncofoetal glycoprotein, 5T4, is strongly expressed by many different cancers but has little expression in normal adult tissues. Its expression is associated with advanced disease and poorer clinical outcome in a number of cancer types, including ovarian cancer. 5T4 is mechanistically associated with the directional movement of cells through the regulation of epithelial mesenchymal transition, facilitation of chemotaxis and modulation of Wnt signaling. The tumour selective expression of 5T4 has prompted the development of several immunotherapies; including a 5T4 antibody– drug conjugate (A1mcMMAF). The latter is a 5T4 humanized monoclonal antibody (A1) linked by sulfydryl-based conjugation to deliver a microtubuledisrupting agent, monomethyl auristan F (MMAF) via a maleimidocaproyl linker. A1mcMMAF has shown potent in vivo activity in a variety of tumour models, with induction of long term regression after the last dose. Here we test activity versus ovarian cancer cells. Methods: In vitro IC50 values of 5T4-ADC in SKOV3 cells expressing 5T4 and 5T4 knock out cells (SKOV3-5T4KO) were determined using MTS assays. Apoptosis and viability were assessed by Apotox-Glo assay. Anti-tumor activity of A1mcMMAF with or without carboplatin chemotherapy against SKOV3luciferase xenografts in NSG mice models was assessed using bioluminescent imaging. Results: A1mcMMAF selectively killed SKOV3 cells with an IC50 20 fold lower than that seen in SKOV3-5T4KO cells. Within the selective range, A1mcMMAF caused apoptosis in SKOV3 but not SKOV3-5T4KO cells. A1mcMMAF (5 mg/kg IP) treatment of SKOV3 xenografts reduced tumour load, delayed growth and significantly increased survival compared to untreated animals. A combination of A1mcMMAF with optimized dosing of carboplatin showed no toxicity and a greater reduction in tumour load and improved survival compared to A1mcMMAF monotherapy. A1mcMMAF plus carboplatin (25 mg/kg IP) showed a median survival increase of 80% compared to carboplatin alone. Conclusions: A1mcMMAF is selective for cells expressing 5T4 and shows potent anti-tumor activity in SKOV3 xenograft models. These data support the use of A1mcMMAF in ovarian cancer treatment potentially in combination with carboplatin. Conflict of interest: Other Substantive Relationships: A1McMMAF was developed by Pfizer Inc and kindly donated for use in this project which was funded by the Wellcome Trust. Puja Sapra is an employee of Pfizer Inc. and owns company’s stock options and/or units. Peter Stern has received honoraria from Pfizer Inc. 648 Membrane Hsp70 as a biomarker for aggressive prostate cancer and therapeutic target G.A. Foulds1 , N. Dunning-Foreman1 , S. Stangl2 , M. Gehrmann2 , J. Vadakekolathu1 , D. Boocock1 , R. Rees1 , G. Multhoff2 , A.G. Pockley1 . 1 Nottingham Trent University, John van Geest Cancer Research Centre, ¨ Munchen, Nottingham, United Kingdom, 2 Technische Universitat ¨ Department of Radiation Oncology, Munich, Germany Introduction: Professor Gabriele Multhoff first identified the selective expression of a membrane form of Hsp70 (memHsp70) on the plasma membrane of tumour cells (but not normal tissue) using a unique monoclonal antibody (mAb, cmHsp70.1, multimmune GmbH). The expression of memHsp70 has since been shown to associate with aggressive disease (e.g. metastasis) and an unfavourable prognosis and poorer survival in patients with cancer. An ongoing screening program of solid tumours at the Technische Universitat ¨ Munchen ¨ is revealing that more than 50% of all tumours express memHsp70.