Affilin

3D structural model of an affilin based on gamma crystalline B (according to PDB  2JDG ).

An affilin is an artificial protein that is capable of binding antigens . Affilins are structurally derived from human ubiquitin or from human gamma crystallins . They are obtained by changing their amino acids close to the surface and isolated with the help of so-called display techniques. Affilins have the properties of antibodies as well as those of low molecular weight substances and are therefore referred to as antibody mimetics . Affilins are currently being investigated and developed as potential novel biopharmaceutical drugs .

structure

Both gamma-crystalline B and ubiquitin have been described as the basic framework for the development of affilins. A binding region within a β-sheet structure is characteristic of both types of affilins . They thus differ from antibodies whose binding region is located in flexible loop regions , the Complementarity Determining Regions (CDRs).

Gamma Crystalline B-based Affilins

Affilins based on gamma-crystalline B are derived from the ocular lens structure protein gamma-crystalline B. This protein with a molecular mass of about 20  kDa consists of two structurally identical protein domains with a β-sheet structure. The eight near-surface amino acids 2, 4, 6, 15, 17, 19, 36 and 38 can be exchanged for others.

Ubiquitin-based affilins

Ubiquitin-based affilins are derived from the ubiquitin protein ubiquitin, which occurs around 76 amino acids in length. This consists of three and a half alpha-helical turns and an anti-parallel β-sheet made up of five strands. The near-surface amino acids 2, 4, 6, 62, 63, 64, 65 and 66 are in spatial proximity to one another at the beginning of the first N-terminal β-sheet strand, in the loop or at the beginning of the C-terminal β-sheet strand and form with their side chains a coherent area on the surface of the ubiquitin. These amino acids can be exchanged for others in the approximately 10 kDa ubiquitin-based affilins.

properties

Affilins have an approximately eight or 16 times smaller molecular weight than antibodies of the IgG type. Based on this, they have an increased tissue permeability compared to antibodies. On the other hand, the low molecular weight enables excretion via the kidneys and leads to a shortened plasma half-life . Their pH stability enables intestinal transit. Affiline are also temperature stable up to 90 ° C.

Manufacturing

The production of affilins against a specific target protein involves several steps. The starting point is the generation of an Affilin molecule library . This affilin library is generated by substituting the eight interchangeable near-surface amino acids of gamma-crystalline B or ubiquitin with the help of random mutagenesis . When using the proteinogenic amino acids with the exception of cysteine , the library can reach a complexity of up to 17 billion different molecules. With a randomization of 14 positions in an Affilindimer, even about 10 ¹⁹ different molecules are conceivable in a dimer library.

After an affilin library has been generated, individual affilins that bind the target protein are selected from it. Display techniques such as phage display or ribosome display are used for the selection . The affilins selected in this way are isolated and further characterized biochemically, pharmacologically and biophysically.

After a selection of affilins, these can be optimized through further changes. Thanks to the avidity effect, dimerization or multimerization can bring about an increase in the binding affinity for the target molecule and at the same time an increase in the plasma half-life. Further options are conjugations with radionuclides , cytotoxins or cytokines .

The large-scale production of affilins can take place with the aid of the production organisms commonly used in biotechnology, such as E. coli .

literature

• Hey T, Fiedler E, Rudolph R, Fiedler M: Artificial, non-antibody binding proteins for pharmaceutical and industrial applications . In: Trends Biotechnol . . 23, No. 10, October 2005, pp. 514-22. doi : 10.1016 / j.tibtech.2005.07.007 . PMID 16054718 .
• Ebersbach H, Fiedler E, Scheuermann T, et al. : Affilin-novel binding molecules based on human gamma-B-crystallin, an all beta-sheet protein . In: J. Mol. Biol. . 372, No. 1, September 2007, pp. 172-85. doi : 10.1016 / j.jmb.2007.06.045 . PMID 17628592 .

Web links

• Website of the affilin manufacturer Scil Proteins GmbH

Individual evidence

1. ↑ Trademark register
2. ↑ Jaenicke R, Slingsby C: Lens crystallins and their microbial homologs: structure, stability, and function . In: Crit. Rev. Biochem. Mol. Biol . 36, No. 5, 2001, pp. 435-99. doi : 10.1080 / 20014091074237 . PMID 11724156 .
3. ↑ Patent WO0104144 : Design of beta-sheet proteins with specific binding properties. Filed on July 13, 2000 , published on 18 January 2001 , Applicant: Scil Proteins GmbH, inventor. Fiedler U., Rudolph R
4. ^ Vijay-Kumar S, Bugg CE, Cook WJ: Structure of ubiquitin refined at 1.8 A resolution . In: J. Mol. Biol. . 194, No. 3, April 1987, pp. 531-44. PMID 3041007 .
5. ↑ Patent WO2006040129 : Protein conjugals for use in therapy, diagnosis and chromatography. Filed on October 11, 2005 , published on April 20, 2006 , Applicant: Scil Proteins GmbH, inventor. Fiedler E., Ebersbach H., T. Hey, Fiedler U