Affilines are artificial proteins which are capable of binding antigens. Affilines are structurally derived from the human ubiquitin or of human gamma - crystallins. They are obtained by changing their near-surface amino acids, and isolated with the aid of so-called display techniques. Affilines possess both properties of antibodies and the therefore called low-molecular substances and are used as antibody mimics. Affilines are currently being investigated as a possible novel biopharmaceutical drugs and developed.
As a basic framework for the development of Affilinen both gamma -crystallin B and ubiquitin have been described. Characteristics of both types of Affilinen is a binding region within a β -sheet structure. They thus differ from antibodies whose binding region is located in flexible loop regions, the complementarity determining regions ( CDRs).
Gamma -crystallin -B -based affilines
Gamma -crystallin -B -based affilines are derived from the eye lens structural protein gamma -crystallin B. This protein with a molecular mass of about 20 kDa consisting of two structurally identical protein domains with β -sheet structure. The eight amino acids close to the surface 2, 4, 6, 15, 17, 19, 36 and 38 can be replaced by others.
Ubiquitin -based affilines
Ubiquitin -based affilines derived from the ubiquitous about 76 amino acids long protein ubiquitin. This consists of three and a half alpha helical windings and a composed of five strands, antiparallel β -sheet. The near-surface amino acids 2, 4, 6, 62, 63, 64, 65 and 66 are in proximity to each other 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 contiguous area on the surface of ubiquitin. These amino acids can be kDa ubiquitin-based Affilinen exchanged in about 10 against others.
Affilines have about eight times or 16 times smaller molecular mass than antibodies of the IgG type. Based on this, they have to antibodies increased tissue permeability. On the other hand enables the low molecular weight and excretion by the kidneys leads to a reduced plasma half-life. Your pH stability allows intestinal transit. Affilines are also stable up to 90 ° C.
The production of Affilinen against a specific target protein involves several steps. The starting point is the generation of a Affilin molecule library. This Affilinbibliothek is generated by substitution of the eight interchangeable near-surface amino acids of gamma -crystallin B or ubiquitin using random mutagenesis. When using proteinogenic amino acid other than cysteine for the library to obtain a complexity of up to 17 billion different molecules. In a randomization of 14 positions in a Affilindimer even about 1019 different molecules in a Dimerbibliothek are conceivable.
After the generation of a Affilinbibliothek be selected from this single affilines which bind the target protein. For the selection will display techniques such as phage display or ribosome display used. The thus selected affilines be isolated and further characterized biochemically, pharmacologically and biophysically.
After selection of Affilinen this can be optimized by further modifications. A di-or multimerization may cause an increase in binding affinity for the target molecule, and at the same time an extension of the plasma half-life due to the avidity. Other options include conjugation with radionuclides, cytotoxins or cytokines.
Large-scale production can be effected with the aid of Affilinen the conventionally used in biotechnology production organisms such as E. coli.