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,
iGEM Team Potsdam Bioware 2012
immunization, killing, cell fusion, selection cloning, reformatting for production in CHO cells >> several months<<
Hybridoma Technique
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Phage Display Technique
selection of fragments
Chinese Hamster Ovary Cells
1. 2. 3. Diverse Antibodies Maturation by Enzyme
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Virus, magnetic beads and FACS for selection
Camelid Antibody Human Antibody Single-Chain Fragment Variable domain (scFv) Single Domain Antibody, Nanobody small antibody construct
scFv425
BBa_K929101
scFv domain anti-EGFR domain 3
4 Fc CH2 CH3 VL CL CH1 VH CH2 CH3 Fc VHH VHH
Human IgG1 CH region
Anti- GFP
BBa_K929107
Nanobody anti-GFP/YFP
advanced antibody construct
Fc
Small Antibody Construct
Human IgG1 CH region
Anti- GFP Fc
BBa_K929107
anti-GFP Nanobody
Signal
TMD
transmembrane domain
TEV TEV protease cleavage site
scFv425
Signal
BBa_K929101
TMD
TEV Signal peptide human EGFR transmembrane domain
TEV protease cleavage site anti-EGFR-D3 scFV 5
Advanced Antibody Construct
Human IgG1 CH region
Anti- GFP Fc
BBa_K929107
anti-GFP Nanobody
Signal
TMD
transmembrane domain
TEV TEV protease cleavage site LoxP LoxP LoxP site LoxP site
Cre
Cre recombinase-DNA
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Advanced Antibody Construct
scFv425
Signal
BBa_K929101
Small Antibody Construct
Transmembrane domain
TEV
Advanced Antibody Construct
eYFP Enhanced YFP ( BBa_E0030) 7 Human IgG1 CH region
Anti- GFP Fc
BBa_K929107
anti-GFP Nanobody
Signal
TMD
TEV LoxP LoxP mCherry transmembrane domain
receptor TEV protease cleavage site
Favorite Biobrick
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
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membrane localization of advanced antibody construct BBa_K929107
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proof of functionality of Cre recombinase
Antibody
Cre
cytidine C A T A A T uridine AID
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Cytidine deaminase motif APOBEC-like C-terminal NES
NLS pSB1C3 Modified AID 11
Cytidine deaminase motif APOBEC-like C-terminal NES
enzyme: AID(activation-induced cytidine deaminase)
NLS eGFP pSB1C3 Modified AID + eGFP
Favorite Biobrick
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nuclear localization of modified AID+eGFP co-transfection of AID and advanced antibody construct
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comparison of mutation rates between wt and modified AID
0.0005 0.001 0.0015 0.002 0.0025
without AID wt AID
mutation/sequenced nucleotides
Fluorescent activated cell sorting (FACS) Magnetic Beads Viral selection Magnetic Beads
GFP
Laser Photomultiplier
+
+ labeled antigen electrical wire
Fluorescent activated cell sorting (FACS)
Laser Photomultiplier
+
+ labeled antigen electrical wire
Viral selection Magnetic Beads
GFP
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Fluorescent activated cell sorting (FACS) Magnetic Beads Viral selection Magnetic Beads
GFP
Laser Photomultiplier
+
+ labeled antigen electrical wire
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antibiotic resistance
Neo- mycin left ITR right ITR CMV
no unspecific infection by rAAV – negative control Target cell line infected with rAAV – positive control
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Assembled with ViralBrick
sortase Virus with sortase motif is completely assembled Expression of EGFR-D3 (~25 kDa)
D3
wt CHO with AID Infected wt CHO Infected mutated CHO 𝑙𝑒𝑒𝑒𝑒𝑒 𝑙𝑒𝑒𝑒𝑒𝑒 𝑙𝑚𝑚𝑚𝑚 𝑙𝑚𝑚𝑚𝑚 𝑙𝑛𝑛𝑒 𝑙𝑒𝑒𝑒 𝑙𝑒𝑒𝑒 𝑙𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑙𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑙𝑒𝑒𝑒𝑗 𝑙𝑒𝑒𝑒𝑗 𝑙𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 𝑙𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒
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Mutated CHO with AID
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initial virus concentration initial wt cell concentration % successful experiments
Chinese Hamster Ovary Cells
1. Antibody library 2. Maturation by Enzyme
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3. Virus, magnetic bead and FACS for selection Stable and transient transfection of both AB constructs Membrane localization Transient transfection
Co-transfection of AID and advanced construct Determination of mutation rates for wt and modified AID Functionality of Cre recombinase Expression of EGFR D3 Preparation and control tests on the viral construct Initial tests on FACS and magnetic beads
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digestion with Apa I and Sph I RFP expression cassette Sph I Apa I Gene of Interest Gene of Interest Gene of Interest PCR with Phosphorothioate Primers Knock out of Thiophosphates with iodine/ethanol solution at 70°C Gene of Interest
s s s s s s s s s s s s PLICing
Blanusa, M., et al. (2010) Anal. Biochem., 406:141
E X S P E X S P
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The concept of SocialBricks is to modularize human practice activities in order to provide adapted versions of activities to various target audiences. People Politics
Minister of Education and Research Prof. Dr. Schavan (CDU)
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Parliamentary State Secretary for Education and Research Dr. Braun (CDU) … and 6
3 scientific conferences in Germany Mobile street lab Survey people about Synthetic Biology Street lab for the Day of Synthetic Biology
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the federal laws
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done by us!
material and lab access.
iGEM Team Potsdam Bioware 2012