vom Fachbereich Chemie der Technischen Universität Darmstadt zur Erlangung des Grades Doctor rerum naturalium (Dr. rer. nat.) Dissertation von Ashi Sapir M. Sc. Erstgutachter: Prof. Dr. Harald Kolmar Zweitgutahter: Prof. Dr. Felix Hausch Darmstadt 2019 Tag der Einreichung: März 2019 Tag der mündlichen Prüfung: Mai 2019 Ashi Sapir: Evaluation of Yeast Display Fab Libraries Derived from Tumor Infiltrating B Cells Darmstadt, Technische Universität Darmstadt, Year thesis published in TUprints: 2020 Date of the viva voce: 27.05.19 Published under CC BY-NC-ND 4.0 International https://creativecommons.org/licenses/ Die vorliegende Arbeit wurde unter der Leitung von Herrn Prof. Dr. Harald Kolmar am Clemens-Schöpf- Institut für Organische Chemie und Biochemie der Technischen Universität Darmstadt sowie bei Merck KGaA in Darmstadt von Januar 2016 bis Januar 2019 angefertigt. Table of contents 1…..ABSTRACT .................................................................................................................................................. 1 1.1. Zusammenfassung .................................................................................................................................... 1 1.2. Abstract .................................................................................................................................................... 3 2.....INTRODUCTION ......................................................................................................................................... 5 2.1. Role of B cells and antibodies in the immune system ..................................................................... 5 2.1.1. Antibody structure and function ......................................................................................................... 6 2.2. Monoclonal antibodies in cancer therapy .......................................................................................... 8 2.3. In vitro antibody selection by display technologies ....................................................................... 10 2.3.1. In vitro antibody selection by yeast display .................................................................................... 11 2.4. Phenotypic screening for antibody discovery, cell-based assay screening ................................. 12 2.4.1. Yeast display biopanning screening ................................................................................................. 14 2.5.1. Tumor infiltrating B Cells (TIL-B) .................................................................................................. 16 2.5.2. TIL-B as an efficient source of highly specific immunoglobulins recognizing tumor membrane proteins ................................................................................................................................................. 16 2.4. Aim of this study ................................................................................................................................. 18 3…..MATERIAL ................................................................................................................................................ 19 3.1. Tissues ................................................................................................................................................... 19 3.2. Bacterial strains, yeast strains and human cell lines ........................................................................ 19 3.3. Plasmids ................................................................................................................................................. 21 3.4. Enzymes and proteins .......................................................................................................................... 25 3.4.1. Antibodies ............................................................................................................................................. 25 3.4. Oligonucleotides .................................................................................................................................. 26 Table of contents I 3.5. Chemicals and supplements ................................................................................................................. 29 3.6. Cell culture media ................................................................................................................................ 31 3.7. Solutions, media and buffer ................................................................................................................ 32 3.8. Kits and laboratory materials............................................................................................................... 32 3.9. Equipment ............................................................................................................................................. 33 3.10. Software ................................................................................................................................................. 34 4…..METHODS ................................................................................................................................................. 35 4.1. Molecular biological methods ............................................................................................................ 35 4.1.1. Total RNA isolation from patients’ tissues ....................................................................................... 35 4.1.2. Determination of DNA/RNA concentration ..................................................................................... 35 4.1.3. Determination of RNA integrity number .......................................................................................... 36 4.1.4. Polymerase chain reaction ................................................................................................................... 36 4.1.5. Reverse transcriptase to synthesize first-strand cDNA ................................................................... 37 4.1.6. Real time PCR for quantitative gene expression analysis ............................................................... 37 4.1.7. Purification of DNA and gel extraction .............................................................................................. 38 4.1.8. Gel electrophoresis .............................................................................................................................. 38 4.1.9. Sanger DNA sequencing ...................................................................................................................... 38 4.1.10. Next-generation sequencing................................................................................................................. 38 4.2. Microbiological methods .................................................................................................................... 38 4.2.1. Transformation and cloning in E. coli ................................................................................................ 38 4.2.2. Extraction of pure plasmid DNA from bacterial cultures ................................................................... 39 4.2.3. Lysis of the yeast cells .......................................................................................................................... 39 4.2.5. Construction of Fab libraries by mating of S. cerevisiae cells ..................................................... 40 Table of contents II 4.2.6. Cultivation, induction of Fab surface expression and storage of S. cerevisiae cells .................... 41 4.2.7. Flow cytometry binding assays using yeast surface display ........................................................... 41 4.2.8. Flow cytometry sorting for isolating high binders using yeast surface display ............................ 42 4.2.9. Bio-Panning on cancer derived cell lines using yeast surface display ........................................... 42 4.4. Cell biological methods ...................................................................................................................... 42 4.4.1. Cultivation of mammalian cells .......................................................................................................... 43 4.4.2. Transfection of mammalian cells and antibody expression ............................................................ 44 4.4.3. Flow cytometry for cellular binding assay .......................................................................................... 44 4.4.4. Fluorescence microscope for cellular binding imaging on slides ....................................................... 45 4.3. Biochemical and Biophysical methods ............................................................................................. 45 4.3.1. Tissue procurement and tissue immunohistochemistry ...................................................................... 45 4.3.2. Determination of protein concentration ............................................................................................. 46 4.3.3. Protein biotinylation ............................................................................................................................ 46 4.3.4. Protein A affinity purification ............................................................................................................. 46 4.3.5. Biolayer interferometry ........................................................................................................................