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Antibodies have provided many useful applications through the years. Therapeutically, antibodies have provided treatments for various diseases such as: cancers, infectious diseases and allergies. Antibodies have also proven to be useful in various immunology and protein detection techniques, such as ELISA. Reference: Nelson PN, Reynolds GM, Waldron EE, Ward E, Giannopoulos K, Murray PG. Demystified …: Monoclonal antibodies. Molecular Pathology. 2000;53(3):111-7. However, the expensiveness and time-consuming part of antibody production entails that research and development of alternatives is needed. We need to think differently.

Peptide aptamers make up one alternative. These combinatorial recognition proteins have been known for over fifteen years Reference: Colas P. The eleven-year switch of peptide aptamers. Journal of biology. 2008;7(1):2.

Colas P, Cohen B, Jessen T, Grishina I, McCoy J, Brent R. Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2. Nature. 1996;380(6574):548-50. and they provide high specificity and strong binding affinity (see table 1).

**Table 1:** Binding affinites for monoclonal antibodies and peptide aptamers.
	Peptide aptamers	Monoclonal antibodies
Affinity (K_D)	10-100 nM	10^-3-100 nM

Peptide aptamers consist of a variable peptide sequence inserted into a protein scaffold and they only bind their targets through this variable peptide loop. Various scaffolds have been used as carriers and conformational stabilizers of peptide aptamers: green fluorescent protein, gluthatione-S-transferase, staphyllococal nuclease and stefin A. However the most frequently used is the bacterial and human thioredoxin. In our project we construct a system for screening peptide aptamers presented in latter one. See figure 1 for a predicted structure.

In order to maintain the combinatorial ability and diverse specificity of the antibodies, the peptide aptamers have a random peptide loop. These are typical from 10 to 20 amino acids long. To construct these combinatorial peptide libraries, twenty (or ten) repeats of the codon NNK, is inserted into the scaffold, where N is A,T, G or C and K is G or C. The latter is to minimize the number of stop codons. If a 20 aa long peptide loop is chosen, you will get 20²⁰=1,05×10²⁶ different peptide combinations. This should give enough diversification to resemble the diversity of antibodies.

Figure 1: A predicted Peptide Aptamer structure using Phyre².

Why did we choose the hTrx-based peptide aptamer as our alternative to the antibody? In contrary to antibodies, peptide aptamers are small monomeric proteins that have been proven to be efficiently expressed and produced in bacteria such as E. coli - especially when presented in a human thioredoxin scaffold. Reference: Borghouts C, Kunz C, Delis N, Groner B. Monomeric recombinant peptide aptamers are required for efficient intracellular uptake and target inhibition. Molecular cancer research : MCR. 2008;6(2):267-81. Because of their high specificity and strong binding affinity, it has been proposed and verified that they can replace antibodies in many methods involving protein detection. Reference: Evans D, Johnson S, Laurenson S, Davies AG, Ko Ferrigno P, Wälti C. Electrical protein detection in cell lysates using high-density peptide-aptamer microarrays. Journal of biology. 2008;7(1):3. Specific peptide aptamers with anticancer and antiviral activity have already been identified. Reference: Li J, Tan S, Chen X, Zhang CY, Zhang Y. Peptide aptamers with biological and therapeutic applications. Current medicinal chemistry. 2011;18(27):4215-22. Even though they are well described and characterized, peptide aptamers are not used to the same extent as antibodies.

For therapeutic reasons , the scaffold for our peptide aptamer should be not immunogenic in humans. By choosing a protein with human origin as a scaffold for the peptide aptamer, like the human thioredoxin, we expected it to be less immunogenic than other potential scaffolds. With an estimated half-life of 100 hours, Reference: Swiss Institute of Bioinformatics. 2015. Available from: http://web.expasy.org/cgi-bin/protparam/protparam1?P10599%402-105. the human thioredoxin provides a stable scaffold and helps prevent the peptide aptamer for premature degradation. This is of course the half-life for the non-recombinant hTrx. The half-life for the recombinant hTrx is probably influenced by the insertion of the random generated peptide sequence.

Peptide aptamers are by far not the only antibody mimetics known. Other affinity proteins have been studied throughout the years - and few even commercialized. Reference: Key Benefits: Affimers have some simple but essential advantages over antibodies: Avacta Life Sciences; [updated 19-08-2015]. Available from: https://www.avactalifesciences.com/key-benefits. However the market is very small. We believe that the hTrx-based peptide aptamer in combination with bacterial-two hybrid screening method provides an easy and fast alternative to antibody production.

Team:SDU-Denmark/Tour23

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