5/24/11. Agenda. Agenda. Identification and sequencing of VHH against ErbB1, ErbB2 and ErbB3 Experimental section Course Immunobiology

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1 Agenda Identification and sequencing of VHH against ErbB1, ErbB2 and ErbB3 Experimental section Course Immunobiology Camelid heavy chain antibodies and VHH Structure Principle library construction Phage display based selection Selection for function and screening Rob Roovers, Alex Klarenbeek & Hans de Haard 25 th of May 2011 Phage display antibody fragments Phage morphology Replication Controlled expression Ab fragments in E coli Experimental setup Expression of antibody fragments Production of phage Agenda Structure of heavy chain antibodies Camelid heavy chain antibodies and VHH Structure Principle library construction Phage display based selection C H 1 C L V H V L V HH Selection for function and screening Phage display antibody fragments Phage morphology Replication Controlled expression Ab fragments in E coli Experimental setup Expression of antibody fragments C H 2 C H 3 Conventional Antibody Heavy and light chains Both chains required for antigen binding and stability C H 2 C H 3 Heavy-Chain Antibody Only heavy chains Full antigen binding capacity and highly stable V HH Ablynx s Nanobody The smallest functional fragment of a naturally occurring single-chain antibody Production of phage 1

2 Identification Ag specific VHH via phage display Phage display based selection on target binding ph shock Selection for function: isolation of antagonistic antibodies Selection for function: isolation of TNFα antagonistic Nanobodies antibody library Analyze phage antibodies Elution TNF-receptor (10 µm) Incubate with immobilized cytokine Washing Elution by competition with excess of receptor BSA (10 µm) Removal unbound phage Immobilized TNF 400 ng 50 ng 10 ng Conclusion: 20-fold enrichment by elution with receptor 2

3 Screening for Lactococcus bacteriophage neutralizing VHH Agenda Camelid heavy chain antibodies and VHH Structure Principle library construction Phage display based selection Selection for function and screening Phage display antibody fragments Phage morphology Replication Controlled expression Ab fragments in E coli Experimental setup Phage morphology Bacteriophage F1 (M13) is filamentous phage with at the tip 3 to 5 copies of minor coat protein piii The major coat protein pviii present in 2,000 to 2,500 copies pvii pix located at the other side of the phage particle Expression of antibody fragments Production of phage Phage biology: replication cycle Bacteriophage F1 (M13) infects E coli by binding to sexpilus (encoded by F episome) via gene 3 Double stranded (RF) DNA genome replicated within cell Single strand DNA packaged into phage particle and released from cells No lytic event, E coli cells survive, but grow slower (plaques) 3

4 Expression of functional Fv in periplasm of E coli Periplasmic expression of Fab in E coli Phage assembly Phage genome Aminoterminal end gene 3 Genome bacteriophage F1 expressed within periplasm of large (6.4 kb) and encodes E coli, which is mediated by its structural and non-structural own leader sequences Compatible with functional expression of antibody fragments, i.e. intramolecular (or canonical) disulfide bridge proteins Gene 3 (minor coat protein) and gene 8 (major coat protein) used for display Origen of replication formed within each responsible for synthesis of immunoglobulin domain single stranded DNA 4

5 Phagemid based display system Monovalent versus polyvalent display Phagemid vectors smaller (4.5 kb) then phage vectors and more efficient for cloning of large libraries These are plasmids (with antibiotic resistance marker) containing f1 ori They only encode gene 3 antibody fusion All other phage proteins (including wt gene 3) supplied by helperphage (carrying different antibiotic resistance marker) Helperphage defective in replication, meaning that produced phage particles mainly contain phagemid Phagemid system used in experimental part Phage vector fd-tet-sfii/noti Q Phagemid vector derivative of phen1, but contains a hybrid gene3 / pelb leader sequence with SfiI/NcoI cloning site FR4 with BstEII site c-myc followed by His6-tag Expression in suppressor strain (TG1) results to incorporation of Gln (Q) at amber stop codon within N-terminus gene III in small fraction of translated mrna; switching to non-suppressor (f.i. TOP F ) gives production of free VHH only Phage vector fd-tet-sfii/noti also used having a SfiI/NcoI site within hybrid geneiii / pelb leader sequence in combination with a NotI site 20 5

6 Agenda Toxicity of antibody fragments Camelid heavy chain antibodies and VHH Structure Principle library construction Phage display based selection Selection for function and screening Phage display antibody fragments Phage morphology Replication Controlled expression Ab fragments in E coli Experimental setup Expression of antibody fragments Production of phage Antibody fragments can be very toxic when expressed in E coli Two phenotypes: Induction of cell lysis Reduction of growth During antibody expression or propagation of phage it is VERY IMPORTANT to grow cells into log phase without disturbing effects of toxicity leading to growth advantage of clones expressing nontoxic fragments Controlled expression by using the Lac promoter A β-galactosidase (LacZ) responsible for conversion of disaccharide Lactose into monosaccharides Galactose and Glucose If no Lactose, then active inducer binds to operator and thereby blocks transcription of LacZ (panel B) By binding of Lactose to inducer prevention of interaction with operator leading to transcription and translation of LacZ (panel A) B Catabolite repression at high Glucose levels At low Glucose levels camp concentrations are high, which binds to Catabolite Activator Protein (CAP) leading to association with CAP region on DNA resulting in high affinity binding of RNA polymerase to promoter region and active transcription of LacZ gene At high Glucose concentration synthesis of camp is decreased, meaning that RNA polymerase binds with low affinity and transcription is inhibited 6

7 Production of antibody fragments in E coli Prepare overnight culture grown in presence of 2% Glucose: NO ANTIBODY PRODUCTION, thus avoiding stability problems with plasmid; can start from a colony taken from an Amp/Glu plate Inoculate 1:100 in fresh medium with Glucose (0.2% according to DeBellis et al or with 2%) At late log phase (OD600 = ) spin down cells (for culture grown in 2% Glucose) and resuspend in medium with the inducer IPTG When using the DeBellis protocol (0.2% Glucose) simply add IPTG; Glucose is consumed and therefore there is no repression of Lac promoter Continue growth and harvest cells Remark: It is important that cell grow rapidly during the log phase (doubling time of 20 to 30 minutes), meaning that with three hours after starting the culture the OD600 should be reached; if this is not the case, then there is something seriously wrong!!! DeBellis publication Repressor Glucose can be included at low concentrations and does not repress during addition of inducer IPTG But if production problems occur due to productions of low levels of very toxic antibody fragments it is recommended to grow with 2% Glucose and spin away Glucose during induction Phage production phagemid libraries A431 and MCF7 Library clonal diversity 10E6-10E8 Implications: Inoculum size Culture size Rescue of library (i.e. helperphage infection) Desired phage number to produce for selection Library + helperphage à Phage (Φ) particles (Phagemid Amp R ) (Kan R ) Production in 2xYT Ampicillin (or Carbenicillin) and Kanamycin NO glucose Phage production in TG1 suppressor strain without inducer VHH/P3 fusion expression by leaky expression Efficient display on phage Phage production of phagemid libraries Experimental procedure Inoculate library in 2xYT(or LB) media with 2% glucose, and ampicillin Starting OD600 of culture preferably <0.05 Grow until early log-phase (OD600=0.5) Infect with helperphage (= rescue) Protocol 1 OD600 unit TG1 ~ 8EXP8 cells 50ml of 2xYT 2% glucose, Ampicillin 100ug/ml in sterile baffled flask Inoculate using 2.5 OD600 units = 2EXP9 cells: Starting OD = 0.05 Grow until OD600 = 0.5 (time is library dependent) Infect 2.5ml (1EXP9 cells) with helperphage 1:1 helperphage:cells results in 50-80% infection 10:1 helperphage:cells results in >95% infection Incubate for (without shaking) Spin culture to remove glucose! 10min. 4,500 x Resuspend cell pellet in 50ml 2xYT medium containing carbenicillin, and kanamycin Grow overnight (ON) in baffled flask for phage production 27 7

8 Phage precipitation and preparation for selection Phage precipitation = purification 2-step procedure Preparation for selection What project? Antigen QC Selection scheme Antigen coating Identification and sequencing of VHH against ErbB1, ErbB2 and ErbB3 Experimental section Course Immunobiology Rob Roovers, Alex Klarenbeek & Hans de Haard 25 th of May