Text The starting point of our project is the creation of a library. A library is a collection of identical plasmids that only vary in the protein coding sequences (CDS). These CDS are designed to possess optimized and planned randomized subregions displaying high variability. The result is a wide variety of plasmids with different inserts. After transformation into E. coli a heterogeneous culture is crated with each colony carrying a different insert translating into different binding proteins (Osoegawa K et al 2001).
To determine possible binding proteins, we compared different types of antibodies and antibody mimetics from various sources . After deep and thorough investigation we decided on using Monobodies and Nanobodies. Due to their small size (10-15k Da) they can outperform regular antibodies (~150kDa) in terms of penetrate depth even into hard tissue However, commonly used antibodies might be more robust. Additionally, nano- and monobody binding proteins are rapidly cleared from the blood and have lower retention time in non-target tissues (Ahmad ZA et al, 2012) .
One advantage of monobodies compared to regular antibodies are the higher stability and the smaller size. Antibody mimetics also show better accessibility to genetic engineering like construction of fusion proteins or incorporation of modifiable amino acids thus allowing the combination with fluorophores or drugs for targeted drug delivery (Skerra et al 2007).
But why using a library? One major advantage of our system is with the availability of a wide range of different binding proteins as starting material for the evolution process. Completely new antibodies or variations of existing ones could serve as starting material.
By creating such a large variety, we are aiming for the highest chance for a possible binder. By adjusting the variable regions and their nucleic acid scheme we can specify the target further but still have leverage to cover a wide range of possible targets in the beginning.

Text Monobodies, also called adnectines , are synthetic antibody mimetics that originate from the tenth human fibronectin type 3 domain. Fibronectin is a rather large protein that is essential in the formation of the extracellular matrix and cell-cell interactions (Koide A, Koide S, 2007). Using its scaffold like an antibody and its possessing similar binding properties was first discovered in 1988 by a research group of Akiko Koide (Koide A et al, December 1998). Since then continuous research lead to appliance in treatment of diseases like glioblastoma multiform (Bloom L, Calabro V, July 2009) or chronic myelogenous leukaemia (Grebien F et al, 2011) Their structure is similar to immunoglobulin domains (Lipovsek, D, 2010). The original three dimensional structure of the scaffold is a β-sandwich but with seven β-strands instead of nine which are common in the variable domain of the heavy chain domain of regular antibodies and three surface loops on each end (Koide, A; Koide, S, 2007)(Koide S, Koide A, Lipovšek D, 2012). They have a high stability and due to their small size, 10kDa, and they penetrate deep into tissue (Koide, A; Koide, S, 2007). Another outstanding feature is that they are functionally expressed in the E. coli cytoplasm and thereby can be produced simple and at low cost in culture. This is due to the absence of disulphide bonds which are unlikely to form in the E. coli cytoplasm . This makes them compatible with virtually any display technologies (Koide A, Koide S, 2007) on the one hand and our in vivo system on the other hand. By randomising the amino acid sequence of the CDR-like loops an initial library can be created. The extensive loop sequence variation is tolerated by the structure so it stays stable (Batori, V. et al. 2002). Thus allowing binding of the target with high affinity and selectivity (Skerra, A, 2007)(Gebauer, M. and Skerra, A,2009)( Gill, D.S. and Damle, N.K, 2006).

Text Nanobodies are single domain antibodies that originate from camelids like Alpaca and Camel or cartilaginous fish like sharks (Stanfield et al., 2004; van der Linden et al., 1999) .
The nanobody family consist of conventional heterotetrameric antibodies with low-affinity binders (Harmsen MM, De Haard HJ, November 2007) as well as unique functional heavy (H)-chain antibodies (HCAbs) with high affinity binders (Harmsen MM, De Haard HJ, November 2007). Because a single polypeptide chain is expressed, they are better suited for phage display than Fragment antigen-binding (Fab) fragments (Holt L. J et al 2003). The H chain of these homodimeric antibodies consists of one antigen-binding domain, the VHH, and two constant domains. HCAbs fail to incorporate light (L) chains owing to the deletion of the first constant domain and a reshaped surface at the VHH side, which normally associates with L chains in conventional antibodies (Muyldermans, S, 2013).
Therefore, the production takes place in liquid cell culture and thus production in large quantities is simple with higher yields than Fab fragments (Holt L. J et al 2003). They consist of a single monomeric variable antibody domain and a universal Nanobody-framework (in our case cAbBCII10). With a size of 12-15 kDa they are slightly bigger than Monobodies but still able to penetrate hard tissues like solid tumours (Saerens, D.; Ghassabeh, G.; Muyldermans, S, 2008). . Research lead to various applications in the therapeutically field. For example, a humanised scFv is in phase II testing to reduce mortality and myocardial infarction in patients undergoing graft surgery for coronary artery bypass (Holt L. J et al, 2003) . Also therapeutically application against the influenza A H5N1 virus has been shown in mice where it reduced the replication rate significantly (Ibanez L. I. et al, 2011). Even in oral immunotherapy Nanobodies are used (Harmsen M. M. et al 2006) . Also many diagnostically applications were made possible like super-resolution microscopy using a Nanobody tagged with GFP (Ries J. et al, 2012) or tagging by binding proteins, enzymes or antigens (Rothbauer U. et al, 2006) with such a complex (Rothbauer U. et al, 2007).They can also be modified to work as a biosensor (Saerens D. et al, 2005).
A high expression rate was shown in E. coli and they are still functional and stable even in absence of conserved disulphide bonds (Saerens et al., 2005) which are unlikely to form in the E. coli cytoplasm. Their high toerance against acidic conditions, temperature (compared to mouse monoclonal antibodies (Van Der Linden R. et al, 1999), high solubility (Arbabi Ghahroudi M. et al, 1997) and very few cleavage sites for enzymes allow new areas of application (Harmsen, M. M.; Haard, H. J. de, 2007) . The Nanobody loop structure can bind to antigen structures inaccessible to human antibodies, for example catalytic centres in enzymes. Also fusion proteins and complexes can be created leaving both parts still active. Example are a complex of a shark Nanobody and lysozyme (Stanfield R. et al, 2004)(Desmyter A. et al, 1996) or a Nanobody conjugated branched Gold nanoparticle for photothermal therapy against tumour cells (Van De Broek B, 2011).

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