Antibody Generation System Maturation, Selection and Production in - PowerPoint PPT Presentation

Antibody Generation System Maturation, Selection and Production in CHO Cells iGEM Team Potsdam Bioware 2012

Antibodies - State of the Art Hybridoma Technique Phage Display Technique immunization, killing, selection of fragments cell fusion, selection cloning, reformatting for production in CHO cells >> several months<< 2

Our Approach 1. 3. 2. Diverse Maturation Antibodies Virus, magnetic by Enzyme beads and FACS for selection C hinese H amster O vary Cells 3

Antibody Formats VH VL Human Antibody Camelid Antibody VHH VHH CH1 CL CH2 Fc CH2 CH3 Fc CH3 S ingle- C hain F ragment Single Domain Antibody, V ariable domain (scFv) Nanobody Nanobody scFv domain anti-EGFR domain 3 Human IgG1 CH region anti-GFP/YFP Anti- scFv425 Fc GFP BBa_K929101 BBa_K929107 small antibody construct advanced antibody construct 4

Antibody Constructs Small Antibody Construct TEV protease transmembrane domain Signal peptide anti-EGFR-D3 cleavage site of B-cell receptor human EGFR scFV Signal TEV TMD scFv425 BBa_K929101 Advanced Antibody Construct TEV protease anti-GFP Nanobody Human IgG1 CH region cleavage site transmembrane domain of B-cell receptor Anti- TEV Signal Fc TMD GFP BBa_K929107 5

Antibody Constructs Cre recombinase-DNA Cre TEV protease LoxP site anti-GFP Nanobody Human IgG1 CH cleavage site transmembrane domain LoxP site region of B-cell receptor Anti- TEV LoxP LoxP Signal Fc TMD GFP BBa_K929107 Advanced Antibody Construct 6

Antibody Constructs Small Antibody Construct Enhanced YFP ( BBa_E0030) Signal TEV Transmembrane domain eYFP scFv425 BBa_K929101 Advanced Antibody Construct TEV protease anti-GFP Human IgG1 CH transmembrane domain cleavage Nanobody region of B-cell site receptor Anti- TEV LoxP TMD mCherry LoxP Signal Fc GFP BBa_K929107 Favorite Biobrick 7

Antibody Results Transient transfection of CHO cells with advanced antibody construct (BBa_K929107) Transient transfection with smaller antibody construct (BBa_K929101) Stable transfection of CHO cells with advanced antibody construct (BBa_K929107) Stable transfection with smaller antibody construct (BBa_K929101) both by application of Flp-In system 8

Antibody Results membrane localization of advanced antibody construct BBa_K929107 proof of functionality of Cre recombinase Antibody Cre 9 9

Mutation Module • enzyme: AID (activation-induced cytidine deaminase) deaminates cytidines in DNA  U:G mismatches  point mutations • cytidine uridine C A T A T A AID 10

Mutation Module  enzyme: AID (activation-induced cytidine deaminase) NLS Cytidine deaminase motif APOBEC-like C-terminal NES pSB1C3 Modified AID 11

Mutation Module Biobrick Design enzyme: AID(activation-induced cytidine deaminase) NLS Cytidine deaminase motif APOBEC-like C-terminal NES eGFP pSB1C3 Modified AID + eGFP Favorite Biobrick co-transfection of AID and advanced antibody construct nuclear localization of modified AID+eGFP 12

Mutation Rate mutation rate: 1. co-transfection of antibody construct & AID -incubation for several days 2. lysis of CHO cells & plasmid preparation 3. retransformation into E. coli, sequencing result (each 5000 bp sequenced): 0.0025 comparison of mutation rates mutation/sequenced nucleotides between wt and modified AID 0.002 0.0015 0.001 0.0005 0 13 without AID wt AID mod. AID mod. AID eGFP

Selection Module Approaches Fluorescent activated cell sorting Magnetic Beads Viral selection (FACS) Cell suspension + labeled antigen Photomultiplier Laser GFP electrical wire - Magnetic - + Beads - - 14

Selection Module Approaches Fluorescent activated cell sorting Magnetic Beads Viral selection (FACS) Cell suspension + labeled antigen Photomultiplier Laser GFP electrical wire - Magnetic - + Beads - - 15

Selection Module Approaches Fluorescent activated cell sorting Magnetic Beads Viral selection (FACS) Cell suspension + labeled antigen Photomultiplier Laser GFP electrical wire - Magnetic - + Beads - - 16

Viral Selection Principle + antibiotic resistance left Neo- right CMV ITR mycin ITR 17

Selection of Anti-GFP nanobody Target cell line infected with rAAV – positive control no unspecific infection by rAAV – negative control Assembled with ViralBrick 18

Universal Virus Modification - D3 sortase Expression of EGFR-D3 (~25 kDa) Virus with sortase motif is completely assembled 19

Modeling 𝑙 𝑒𝑒𝑒𝑒𝑒 𝑙 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑙 𝑚𝑚𝑚𝑚 Infected wt CHO wt CHO with AID 𝑙 𝑒𝑒𝑒 𝑙 𝑒𝑒𝑒𝑗 𝑙 𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑙 𝑛𝑛𝑒 𝑙 𝑒𝑒𝑒𝑗 𝑙 𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑙 𝑒𝑒𝑒 Mutated Infected CHO mutated CHO with AID 𝑙 𝑚𝑚𝑚𝑚 𝑙 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑙 𝑒𝑒𝑒𝑒𝑒 20

Modeling - Deterministic 21

Modeling - Stochastic % successful experiments initial virus concentration initial wt cell concentration 22

Summary – Antibody System Transient transfection Stable and transient 1. 3. 2. Expression of EGFR D3 of AID transfection of Co-transfection of AID both AB constructs Preparation and control and advanced construct tests on the viral Membrane localization construct Determination of Initial tests on FACS and Antibody library mutation rates for wt Maturation magnetic beads and modified AID Virus, magnetic by Enzyme bead and FACS for selection Functionality of Cre recombinase C hinese H amster O vary Cells 23

Potsdam Cloning Standard PLICing Blanusa, M., et al. (2010) Anal. Biochem. , 406:141 s s s Apa I Sph I E X S P PCR with Gene of Interest RFP expression cassette Phosphorothioate s Primers s s s s s digestion with Gene of Interest Knock out of Apa I and Sph I Thiophosphates with E X S P s s s iodine/ethanol solution at 70°C Gene of Interest Gene of Interest 24

SocialBricks The concept of SocialBricks is to modularize human practice activities in order to provide adapted versions of activities to various target audiences. Politics People 25

Science meets Politics … and 6 others Minister of Education and Parliamentary State Secretary for Research Prof. Dr. Schavan (CDU) Education and Research Dr. Braun (CDU) 26

Science meets People 27 Street lab for the Day of Synthetic Biology Survey people about Synthetic Biology 3 scientific conferences in Germany Mobile street lab

Software Safety Attribution • Biosafety level 1 • All lab experiments were organisms and plasmids done by us! only We have to thank for • Our laboratory follows all • material and lab access. the federal laws • For details, see our Wiki or poster Sponsoring 28

Thank you for your attention Questions??? iGEM Team Potsdam Bioware 2012