Microbodies™ are protein therapeutics and tools for diagnostic and technical applications

sMall biologicals froM Nascacell

Aptamers are small, functional oligonucleotides with a defined 3D structure and are selected to bind target proteins with affinities and specificities only matched by antibodies. Because of their drug like binding characteristics, aptamers typically show strong inhibition of protein function. Aptamers, either RNA, stabilized RNA or DNA, are selected in an automated procedure from combinatorial nucleic acid libraries of 1015 molecules by an iterative in vitro selection process with proven track record in

• Target validation: Highly specific inhibition and modification of both intra- and extracellular targets through specific binding, in enzyme assays, cellular assays and even in animal models.

• Drug screening: Aptamers can be easily modified and adapted for most available readout systems in HTS systems used today.

Aptamer selected on activated Erk2 kinase displays high affinity for the activated enzyme, yet significantly less affinity for the non-activated enzyme. It is a good example for the selectivity aptamers display for structural changes, a feature that differentiates them from most antibodies (top). Once selected on a target, aptamers can be used to validate a target in cellular assays or in vivo and as competitive ligands in HTS (below). Transfection with the Erk2 kinase aptamers for example inhibits the growth of Erk2 dependent cell lines.

Microbodies™ (green) are ≥ 3 kDa in size and composed of a rigid protein scaffold, stabilized by 3 cystin bonds (yellow). The loop region can be replaced by different target binding peptides (red). A Microbody™, higly specific for β -tryptase (Ki 1-2 nM) has been modelled into one subunit of this protease.

Microbodies™ combine affinity and selectivity of for example antibodies with the ease of handling, stability and storage of small molecule drug compounds. They are very small (28 to 45 amino acids), highly soluble proteins with pseudo-knot structures, which provide excellent stability for this protein class. NascaCell‘s Microbody™ scaffolds allow changes of loop sequences, into which biologically active peptide sequences (5-20 amino acids) are grafted. Alternatively, novel loop-sequences can be selected using bacterial display.

Microbodies™ are stable “sMall biologicals”Microbodies™ have shown therapeutic efficacy in vivo. Protease resistance and a shelf life of almost years at ambient temperatures make them interesting for assay development (e.g. immunology, SPR, competition assays…) and as biological reagents (e.g. diagnostics, affinity chromatography, enzyme inhibition…).

aptaMers as tools in target validation and drug discovery

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Target validation in animal models

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sMall biologicals froM Nascacell

NascaCell Technologies AGMax-Lebsche-Platz 31D-81377 München, Germany

Tel.: +49 (0) 89 54 72 72 0Fax: +49 (0) 89 54 72 72 22Email: customer @ nascacell.de

application notes with exaMples on our web site:www.nascacell.com

2/2008

exaMple: aptaMers specifically binding a MeMbrane protein

Using a special selection strategy NascaCell offers the generation of aptamers specific for membrane proteins. Counter selection on the non-expressing cell line is followed by selection on a cell line expressing the target. Optionally, specific elution with a target binding ligand can be integrated into the procedure as was the case for membrane protein (RecX). The resulting aptamers exhibit affinities for this in the low nanomolar range and bind selectively to cells carrying the receptor protein.

rapid access to a novel tool in early drug discovery

Time is what counts and NascaCell completes projects with automated selection providing 2 characterised aptamers in 8-12 weeks for soluble targets. This means rapid access to a tool that can be synthesized in large quantities and easily labelled for many different applications.

SELEX™ on cells (left). Cellular binding studies: Binding of fluorescently labelled aptamer to RecX expressing and non-expressing cells lines (right).

wild type cells cells expressing RecX

aptamer library

anti-RecX-aptamer