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