Isolation of Optimal Peptide Substrates for a Protease using phage display

protease

column

substrate phage

sequencingbiochemical

analysis

Single chain antibody (ScFv)

+ • generally high levels of expression • relative stability of assembled

protein

-• sometimes fails to reproduce the

activity of natural antibody • potential immunogenicity

Recombinant antibodies for vaccines

Fab fragment +• lower immunogenicity • higher stability in bloodstream • efficiently emulates the activity

of natural antibody -• difficulties in assembly• lower yield of stable protein

VlCl

VhCh1Vl

Vh

Antibodies for medicine

Treatment

Prophylaxis Diagnostics

VlCl

VhCh1

Fab-fragment

Vl

Vh

ScFv

Native antibody

Native antibody

VlCl

VhCh1

Fab-fragment

Vl

Vh

ScFv

Native antibody

Recombinant antibodies: ScFv and Fab fragment

Vl

Cl

Vh

Ch

1V

l

Vh

ScFv

Fab-fragmentFab

Fc

Comparison of hybridoma and phage display approach

Hybridoma technology:• provides isolation of full-length

antibodies specifically interacting with antigen

• only small number of antigen-specific antibody clones can be selected (due to limited starting repertoire)

• antibodies raised as hybridomas are not suitable for human vaccines (murine antibodies)

• large amount of specific antibody can be easily produced (suitable only for detection)

• antibodies require humanization in order to be used for prophylaxis or treatment

Phage display technology:• provides highly specific

recombinant antibodies (Kd up to 10-12)

• broad starting repertoire permits selection of vast number of highly specific antibodies

• large amounts of recombinant antibody can be produced in bacteria

• recombinant antibodies can be used for detection, prophylaxis, and treatment as well

• Bacterial expression of recombinant antibody therapeutic is relatively inexpensive

Our approach to improve stability and achieve correct assembly of Fab fragment

Phage particle Phage particle carrying carrying

initial library of initial library of Fab fragmentsFab fragments

Immobilized anti-FabImmobilized anti-Fab antbodyantbody

AnalysAnalysisis of p of phagehagemidmidss, ,

carrcarryying geneing genes encodings encoding

forfor stable Fab-fragment stable Fab-fragment

Purification of phage

Purification of display phages by gel filtration on Sephacryl S500

Typical chromatographic profile of phage purification

Protein 8,6 kDa

DNA-shufflingCDRIIICDRIICDRICDRIIICDRIICDRI

CDRIIICDRIICDRI

CDRIIICDRII

CDRIII

FRIFRI FRIIFRII FRIIFRIIII

FRIVFRIVCDRI CDRII CDRIII

hg

FRIFRI FRIIFRII FRIIFRIIII

FRIVFRIV

CDRI CDRII CDRIII

CDRIIICDRIICDRI

CDRII

CDRI

CDRI

CDRI CDRIICDRIII

CDRICDRII CDRIII

CDRICDRI CDRII

CDRII CDRIIICDRIII

CDRICDRI CDRIII

CDRIII

CDRIICDRII

ASYMMETRIC ASYMMETRIC PCRPCR

ASYMMETRIC ASYMMETRIC PCRPCR

PCR

Annealing of products

Phage display strategy

Production Production of phage of phage particles particles

Expression of Expression of chimeric geneIIIchimeric geneIII ++

phage proteinsphage proteins

Phage display strategy

AnalysAnalysisis of p of phagehagemidmidss, carr, carryying ing

genegenes encodings encoding for protein of for protein of

interest.interest.

Structure of filamentous phage for display of foreign proteins

GeneIX protein

GeneVII protein

ss DNA

GeneIII protein

GeneVI protein

GeneVIII protein

geneIII protein geneVIII protein

Phages carrying surface proteins, fused with

Phage display-derived antibodies for detection and therapy

Object Publication

VirusesPotato-virus-Y potyvirusHuman HCMVCytokinesHuman interleukin-6HormonesSteroidsOestradiol Growth factor receptorsvEFG receptor-2 (Flk1/Kdr)Tissue and tumor specific markersMUC1 core peptide (adenocarcinoma)Tumor tissue sectionsc-erbB-2 (oncogene product overexpressed by breast carcinomas and other adenocarcinomas)carcinoembryonic antigen (CEA)ANTHRAX

Boonham N., et al, J. Virol. Methods (1998) 74:193-199Takekoshi M., et al, J. Virol Methods (1998) 74: 89-98

Krebs B., et al, J. Biol. Chem. (1998) 273: 2858-2865

Dorsam H., et al, FEBS Lett. (1997) 414: 7-13Vaughan T.J., et al, Nat. Biotechnol. (1996) 14: 309-314

Witte L., et al, Cancer Metastasis Rev. (1998) 17: 155-161

Henderikx P., et al, Cancer Res. (1998) 58: 4324-4332

Tordsson J., et.al, J. Immunol. Methods (1997) 210: 11-23Schier R., et al, Immunotechnology (1995) 1:73-81

Osbourn J.K., et al, Immunotechnology (1996) 2; 181-196Crino N.M., et al, Infection and Immunity (1999) 67:2957-2963Maynard J.A., et al, Nat. Biotechnology (2002) 20: 597-601

Vector for antibody display

Hydrolysis of natural and phage-selected t-PA targets by trypsin and by t-PA

Substrate Enzyme kcat Km kcat /Km Ratio

SPGR↓VVGGS*

SPGR↓VVGGS*

PFGR↓SALVPE#

PFGR↓SALVPE#

PLASMINOGEN

t-PA

Tn

t-PA

Tn

t-PA

0.0043

25

4.2

220

0.1

15000

790

3100

103

6.5

0.29

3.2x10(e4)

1350

2.1x10(e6)

1.5x10(e4)

1

110000

4700

7.2x10(e7)

52000

*natural peptide

#phage-selected peptide

Kinetics of cleavage of natural and phage-selected substrates of plasmin

Substrate Enzyme kcat Km kcat /Km Ratio

SPGR↓VVGGSVA*

LGSGIYR↓SRSLE#

NATIVE MICRO-PLG

MPLG WITH PHAGE

SUBSTRATE

PL

PL

PL

PL

0.0086

120

0.0016

1.5

5100

100

4.7

2.0

1.7

1.2x10(e6)

340

7.1x10(e5)

1

710000

200

417000

*natural peptide

#phage-selected peptide

Substrate phage vector

Tac promoter rbs rrnB

ColE1 oribla

gene III leader c-myc

tagrandom octapeptide gene III