Isolation of Optimal Peptide Substrates for a Protease using phage display protease column substrate phage sequencing biochemical 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 Vl Cl Vh Ch1 Vl Vh

Antibodies for medicine Treatme nt Prophylaxis Diagnostics Vl Cl Vh Ch1 Fab-fragment Vl Vh ScFv Native antibody Vl Cl Vh Ch1 Fab-fragment Vl Vh ScFv Native antibody

Recombinant antibodies: ScFv and Fab fragment Vl Cl Vh Ch1 Vl Vh ScFv Fab-fragment Fab 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 ) 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 carrying initial library of Fab fragments Immobilized anti-Fab antbody antbody Analysis of phagemids, carrying genes encoding for 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-shuffling CDRIII CDRII CDRI CDRIII CDRII CDRI CDRIII CDRIICDRI CDRIII CDRII CDRIII FRI FRIIFRIIIFRIV CDRI CDRIICDRIII hg FRIFRIIFRIIIFRIV CDRI CDRII CDRIII CDRII CDRI CDRII CDRI CDRII CDRIII CDRI CDRII CDRIII CDRI CDRII CDRIII CDRI CDRIII CDRII ASYMMETRIC PCR PCR Annealing of products

Phage display strategy Production of phage particles Expression of chimeric geneIII + phage proteins

Phage display strategy Analysis of phagemids, carrying genes encoding for protein of 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 ObjectPublication Viruses Potato-virus-Y potyvirus Human HCMV Cytokines Human interleukin-6 Hormones Steroids Oestradiol Growth factor receptors vEFG receptor-2 (Flk1/Kdr) Tissue and tumor specific markers MUC1 core peptide (adenocarcinoma) Tumor tissue sections c-erbB-2 (oncogene product overexpressed by breast carcinomas and other adenocarcinomas) carcinoembryonic antigen (CEA) ANTHRAX Boonham N., et al, J. Virol. Methods (1998) 74: Takekoshi M., et al, J. Virol Methods (1998) 74: Krebs B., et al, J. Biol. Chem. (1998) 273: Dorsam H., et al, FEBS Lett. (1997) 414: 7-13 Vaughan T.J., et al, Nat. Biotechnol. (1996) 14: Witte L., et al, Cancer Metastasis Rev. (1998) 17: Henderikx P., et al, Cancer Res. (1998) 58: Tordsson J., et.al, J. Immunol. Methods (1997) 210: Schier R., et al, Immunotechnology (1995) 1:73-81 Osbourn J.K., et al, Immunotechnology (1996) 2; Crino N.M., et al, Infection and Immunity (1999) 67: Maynard J.A., et al, Nat. Biotechnology (2002) 20:

Vector for antibody display

Hydrolysis of natural and phage-selected t- PA targets by trypsin and by t-PA SubstrateEnzymek cat KmKm k cat /K m Ratio SPGRVVGGS* PFGRSALVPE# PLASMINOGEN t-PA Tn t-PA Tn t-PA x10(e4) x10(e6) 1.5x10(e4) x10(e7) *natural peptide #phage-selected peptide

Kinetics of cleavage of natural and phage-selected substrates of plasmin SubstrateEnzymek cat KmKm k cat /K m Ratio SPGRVVGGSVA* LGSGIYRSRSLE# NATIVE MICRO-PLG MPLG WITH PHAGE SUBSTRATE PL x10(e6) x10(e5) *natural peptide #phage-selected peptide

Substrate phage vector Tac promoter rbs rrnB ColE1 ori bla gene III leader c-myc tag random octapeptide gene III