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2 Tabriz University of Medical Sciences Faculty of Advanced Medical Sciences Isolation and Characterization of Human Antibody Fragments to Differentiated Gastric Cancer Cells from a Semisynthetic Phage Display Library Written and presented by Sepideh Nikfarjam Supervisors Prof. Mohammad Nouri Dr. Mohammad Reza Tohidkia Advisor Dr. Ali Akbar Rahim Rahimi July 2019
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Introduction Methodology Results Discussion Conclusion Prospects CONTENTS
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01 Introduction Phage Display Technology Proteomics Monoclonal Antibodies
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Gastric Cancer 5 The stomach: a J-shaped organ in the upper abdomen, a part of the digestive system. In gastric cancer malignant cells form in the lining of the stomach. The fourth most common malignancy worldwide and 90% of cases are adenocarcinomas (Siegel, Miller et al. 2017). Poor clinical outcomes related to scarcity of suitable tumor antigen(s) for early diagnosis and therapy (Heitner, Moor et al. 2010). Comprehensive knowledge about the molecular context of cancerous cells for identification of novel targets and therapeutics. https://www.cancer.gov/types/stomach/patient/stomach-treatment-pdq
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Lauren’s Histopathological Classification of Gastric Adenocarcinomas 6 Well differentiated (AGS) Nonmetastatic M:F 2:1 5 year survival rate 20% Etiology: Blood group A, H. pylori infection, Unknown Poorly differentiated (MKN-45) Metastatic M:F 1:1 5 year survival rate <10% Etiology: Dietary factors, H. pylori infection Copy paste fonts. Choose the only option to retain text. 1) Intestinal 2) Diffuse (Kim, Kang et al. 2011)
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Phage Display Technology 7 Generation of monoclonal antibodies by in vitro selection process termed as ‘panning’. Pioneered by George Smith using filamentous phage genome (Smith 1985). Fully human antibodies against self-antigens such as tumor cell biomarkers (Skerra and Pluckthun 1988). Whole cell panning: relevant binders recognizing naturally exposed epitopes (Lipes, Chen et al. 2008). The integration with proteomics: identification of disease-related biomarkers and discovery of therapeutic antibodies (Hust and Dübel 2004). (Hoogenboom, de Bruıne et al. 2014)
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Objective and Hypotheses of the Study 8 To isolate and characterize human antibody fragments to cell surface proteins of differentiated AGS cells from a semisynthetic phage-scFv library Is it possible to isolate specific phage-scFvs against AGS cell surface proteins from Tomlinson I library? Is there enough diversity among the selected clones? Do the selected clones bind strongly to AGS cells? Are these selected clones capable of discriminating AGS cells from poorly differentiated MKN-45 cells and NIH-3T3 fibroblasts? 02 03 04 Q2 Q3 Q4 Hypotheses 01 Q1 Objective
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02 Methodology Phage Display Technology Proteomics Monoclonal Antibodies
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Methodology 10
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03 Results Phage Display Technology Proteomics Monoclonal Antibodies
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Subtractive Whole Cell Panning 12 Table 1. Details of the Panning Process 95%
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Subtractive Whole Cell Panning (cont.) 13 95% Figure 1. Polyclonal Phage ELISA
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PCR Analysis of the Selected Clones 15 (A) Round 3 (B) Round 4 Figure 3
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Sequence Diversity of the ELISA-positive Clones 16 Table 2
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Sequence Diversity of the ELISA-positive Clones (cont.) 17 Figure 4
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Specificity Characterization of the Clones by Cell-based ELISA 18 Figure 5
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Flow Cytometric Analysis of Phage-scFv Binding 19 Figure 6 (A) AGS MKN-45 NIH-3T3 H1 D11 G11 D1 Negative Control
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Flow Cytometric Analysis of Phage-scFv Binding (cont.) 20 Figure 6 (B)
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04 Discussion Phage Display Technology Proteomics Monoclonal Antibodies
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Discussion 22 Decoding the human proteome: discovering novel biomarkers with a critical role in diseased conditions (Valk et al. 2004). Affinity-based approaches for proteome profiling: utilizing affinity molecules as protein-specific probes to discern and quantify molecular targets in their natural milieu (Poetz et al. 2009). Surface proteome profiling based on monoclonal antibodies: recognizing molecular modifications of the proteins overexpressed in malignancies (Poetz et al. 2009). The application of phage antibody libraries and subtractive whole cell panning strategy: isolation of functional antibodies with higher specificity (Cattaneo 2010). The ability to distinguish between the proteome of different cells and tissues, i.e. healthy and malignant cells (Cattaneo 2010). Antibody arraying and parallel screening on diverse cells/tissues: identification of differentially expressed proteins (Blažek and Celer 2003).
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Discussion 23 Similar investigations: Direct whole cell panning method to screen Tomlinson library against ovarian cancer cells. Proper discriminative reactivity of the clones for the target cells, normal ovarian cell line, and a panel of tumors (Sharma et al. 2007). A subtractive whole cell panning to screen a single domain antibody library against lung adenocarcinoma cells and identifying the cognate antigens (Zhang et al. 2009). Selecting binding and internalizing receptor-specific antibodies from a large naïve scFv library by subtractive whole cell panning against breast cancer cells. 40% of clones bound to the tumor cells but not to normal human cells and the target antigens were identified (Poul et al. 2011). Mapping the variation of gene expression between two populations of cells.
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05 Conclusion Phage Display Technology Proteomics Monoclonal Antibodies
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Conclusion 25 Monoclonal Antibodies Phage Display Technology Proteomics Comparative surface proteomic analysis of differentiated and poorly differentiated gastric adenocarcinoma cell lines. Recognition and characterization of the respective differentially expressed cell surface proteins. Targeted delivery of bioactive moieties such as drugs, toxins, cytokines, and radionuclides to cancer sites.
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06 Prospects Phage Display Technology Proteomics Monoclonal Antibodies
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Early diagnosis; tumor profiling; patient stratification; predicting relapses and survival; monitoring of therapy. Biomarker Discovery New avenues for gene therapy of gastric cancer. Genetic Manipulation of the Isolated Leads The instant availability of antibody-encoding genes can be used for gene function knockdowns. Gene Knockdowns Generation of a renewable resource of 30,000+ monoclonal antibodies to the entire human proteome. Whole Proteome Profiling Prospects 27
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Acknowledgments 28 Miss Mehdipour All the members of staff at Research Center for Pharmaceutical Nanotechnology Department of Medical Biotechnology Members of Faculty of Advanced Medical Sciences My family for their continued support and encouragement. All postgraduate students at Tabriz University of Medical Sciences I would like to thank my supervisors Prof. Nouri and Dr. Tohidkia for providing me an opportunity to complete my thesis, and for their patient guidance and encouragement. My advisor, Dr. Rahim Rahimi