2006

1TrademarksAffibody is a trademark owned and used by Affibody AB. Affibody is a registered trademark in Sweden, EU and USA. Trademark application is pending in Japan.

Affibody® Molecules – A Robust Scaffold

Engineered on a three-helix bundle Protein A domain scaffold

Small (58 aa, 6 kDa)

Stable (temperature, pH, proteases)

Simple structure (no S-S)

Highly soluble

Bacterial production or peptide synthesis

Ig-binding domains of Protein A

E AD CB

Affibody® Molecule Diversity

Randomization of 13 selected positions

Protein A-domainscaffold

3x109 Affibody®

library members

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Nanomolar Affinities to a Variety of Targets

n Target origin• Human proteins

TNFα: <1 nMHER2: 22 pM

• Viral proteinsgp120: 30–70 nM

• Bacterial proteinsTaq Polymerase: 25 nM

n Target size• From 4 to 800 kDa One Target Protein

- Different Binding Properties

Flexibility – Engineering of Affibody® Molecules

n

α-X α-Y

SH

Multimeric

Bi-specificSite-specific modifications

Fusion protein

Biotechnology Applications

ELISA

Affibody® molecule as the capture reagentEasily coated on plasticsWorks well in pair with antibodySensitivity in the pg/ml range

HRP HRP

Immunoprecipitation

Affibody® molecule as capture reagent on solid phase - pull out desired molecule from a complex mixSingle step precipitationNo interference by the 50 kDa heavy and 25 kDa light chains of an immuno-precipitating antibody in the Western blot

n1 bead

Conjugated Affibody®

molecule as ”one step reagent”Unique C-terminal cysteine for directed modification

• Fluorescence labeling• Biotinylation• HRP conjugation

Low background staining and selective binding

Immunofluorescence Cell and Tissue Staining

Fluorescein conjugatedAnti-HER2 Affibody®

molecule staining HER2 expressing human SKOV-3 cell line

HRP conjugatedAnti-HER2 Affibody®

molecule staining tumor tissue

Fluorescein conjugatedAnti-HER2 Affibody®

molecule staining tumor tissue

Immunohistochemistry - Xenograft TumorsHER-2 expression in frozen tissue sections of SK-OV-3 xenograft tumors

Immunohistochemistry – Paraffin Embedded Tissue Sections

HRP conjugated Anti-Insulin Affibody® molecule staining staining pparaffin embedded normal human pancreas

HRP conjugatedAffibody® molecule as convenient ”one step reagent”Low background staining and selective binding

Flow Cytometry

Red line: Oregon® Green-conjugated Anti-EGFR Affibody® moleculeBlack: Negative control

Robust alternative to antibodies

Protein Microarrays

Affibody® molecule as capture reagentEasily coated on most types of commercially available slides for proteins

Affinity Separation

n Isolation of pure target protein

n Removal of the target protein

n Recovery of the flow through fraction for further analysis

Depletion of HSA from Serum

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Overlay chromatograms of repeated affinity removal of HSA from serum. The chromatograms represent run number 1, 50, and 300 after consecutive injections of 300 µl of five times diluted human serum on an 0.37 ml Anti-HSA Affibody® column.

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SDS-PAGE analysis of flow-through fractions (FT) and eluted fractions after repeated affinity removal of HSA from serum. Lane 1: Untreated 5x diluted serum sample; lane 2: FT run 1; lane 3: FT run 75; lane 4: FT run 150; lane 5: FT run 225; lane 6: FT run 300; lane 7: eluate run 1; lane 8: eluate run 300;lane 9: HSA standard.

n Extreme stability• Reversible folding

n High specificity• Binding surface large fraction of total surface

n Engineering flexibilityn Cost-efficient production

• Bacterial expression or chemical synthesis

n Unique patent position• No overlap with conventional aptamer,

monoclonal and domain antibody patents

Affibody® Molecules - Key Competitive Features