5/24/11

1

Identification and sequencing of VHH against ErbB1, ErbB2 and ErbB3

Experimental section Course Immunobiology

Rob Roovers, Alex Klarenbeek & Hans de Haard

25th of May 2011

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

•  Expression of antibody fragments

•  Production of phage

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

•  Expression of antibody fragments

•  Production of phage

Structure of heavy chain antibodies

Combining benefits of antibodies and small chemicals

VH

VLCL

CH1

CH3

CH2

VHH

CH3

CH2VHH

Conventional Antibody

Heavy and light chainsBoth chains required for antigen

binding and stability

Heavy-Chain Antibody

Only heavy chainsFull antigen binding capacity

and highly stable

Ablynx’s Nanobody®®

The smallest functional fragment of a naturally occurring single-chain

antibody

5/24/11

2

Identification Ag specific VHH via phage display

pH shock

Phage display based selection on target binding

Selection for function: isolation of antagonistic antibodies

antibodylibrary

Removal unbound phage

Analyze phage antibodies

Elution by competitionwith excess of receptor

Incubate with immobilized cytokineWashing

antibodylibrary

Removal unbound phage

Analyze phage antibodies

Elution by competitionwith excess of receptor

Incubate with immobilized cytokineWashing

400 ng 50 ng 10 ng

TNF-receptor (10 µM)

BSA (10 µM)

Elution

• Conclusion: 20-fold enrichment by elution with receptor

Immobilized TNF

Selection for function: isolation of TNFα antagonistic Nanobodies

5/24/11

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

•  Expression of antibody fragments

•  Production of phage

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

•  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)

Phage biology: replication cycle

5/24/11

4

Expression of functional Fv in periplasm of E coli

Periplasmic expression of Fab in E coli

Phage assembly

•  Aminoterminal end gene 3

expressed within periplasm of

E coli, which is mediated by its

own leader sequences

•  Compatible with functional

expression of antibody

fragments, i.e. intramolecular

(or canonical) disulfide bridge

formed within each

immunoglobulin domain

•  Genome bacteriophage F1

large (6.4 kB) and encodes

structural and non-structural

proteins

•  Gene 3 (minor coat protein)

and gene 8 (major coat

protein) used for display

•  Origen of replication

responsible for synthesis of

single stranded DNA

Phage genome

5/24/11

5

Phagemid based display system

•  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

Monovalent versus polyvalent display

Phagemid system used in experimental part

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

20

Phage vector fd-tet-SfiI/NotI

•  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

5/24/11

6

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

•  Expression of antibody fragments

•  Production of phage

Toxicity of antibody fragments

•  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 non-toxic fragments

Controlled expression by using the Lac promoter

•  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)

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

5/24/11

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 = 0.8 – 1.0) 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

27

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

•  Phage production in TG1 suppressor strain without inducer •  VHH/P3 fusion expression by leaky expression •  Efficient display on phage

Library + helperphage à Phage (Φ) particles

(Phagemid AmpR) (KanR)

Production in 2xYT Ampicillin (or Carbenicillin) and Kanamycin NO glucose

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 30min. @37C (without shaking) •  Spin culture to remove… glucose! 10min. 4,500 x g @RT •  Resuspend cell pellet in 50ml 2xYT medium containing carbenicillin, and

kanamycin •  Grow overnight (ON) shaking @37C in baffled flask for phage production

5/24/11

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

25th of May 2011